Prevention and treatment of synucleinopathic and amyloidogenic disease

ABSTRACT

The invention provides improved agents and methods for treatment of diseases associated with synucleinopathic diseases, including Lewy bodies of alpha-synuclein in the brain of a patient. Such methods entail administering agents that induce a beneficial immunogenic response against the Lewy body. The methods are particularly useful prophylactic and therapeutic treatment of Parkinson&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 16/282,205filed Feb. 21, 2019, which is a continuation of U.S. Ser. No. 15/608,737filed May 30, 2017, which is a continuation of U.S. application Ser. No.14/841,501 filed Aug. 31, 2015, which is a continuation of U.S.application Ser. No. 13/402,848 filed Feb. 22, 2012, which is acontinuation of U.S. application Ser. No. 12/528,439 filed Dec. 7, 2009now U.S. Pat. No. 8,147,833 issued Apr. 3, 2012, which is a US NationalStage Entry of PCT/US2008/002392 filed Feb. 22, 2008, which claims thebenefit under 35 U.S.C. § 119(e) of US Application Nos. 60/984,721 filedNov. 1, 2007. PCT/US2008/002392 filed Feb. 22, 2008 is also acontinuation-in-part of U.S. application Ser. No. 11/697,646 filed Apr.6, 2007, which is a continuation-in-part of U.S. application Ser. No.11/710,248 filed Feb. 23, 2007 now U.S. Pat. No. 9,034,337 issued May19, 2015, which is a continuation-in-part of U.S. application Ser. No.11/660,015 filed Feb. 9, 2007, which is a US National Stage Entry ofPCT/US2005/028166 filed Aug. 9, 2005, which is a continuation-in-part ofU.S. application Ser. No. 11/185,907 filed Jul. 19, 2005 now U.S. Pat.No. 8,506,959 issued Aug. 13, 2013. Each of these applications isincorporated by reference in its entirety for all purposes.

SEQUENCE LISTING

This application includes an electronic sequence listing in a file named547065SEQLST.txt, created Oct. 23, 2020 and containing 23,109 bytes,which is hereby incorporated by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

Alpha-synuclein (alpha-SN) brain pathology is a conspicuous feature ofseveral neurodegenerative diseases, including Parkinson's disease (PD),dementia with Lewy bodies (DLB), the Lewy body variant of Alzheimer'sdisease (LBVAD), multiple systems atrophy (MSA), and neurodegenerationwith brain iron accumulation type-1 (NBIA-1). Common to all of thesediseases, termed synucleinopathies, are proteinaceous insolubleinclusions in the neurons and the glia which are composed primarily ofalphaSN.

Lewy bodies and Lewy neurites are intraneuronal inclusions which arecomposed primarily of alpha-SN. Lewy bodies and Lewy neurites are theneuropathological hallmarks Parkinson's disease (PD). PD and othersynucleinopathic diseases have been collectively referred to as Lewybody disease (LBD). LBD is characterized by degeneration of thedopaminergic system, motor alterations, cognitive impairment, andformation of Lewy bodies (LBs). (McKeith et al., Clinical andpathological diagnosis of dementia with Lewy bodies (DLB): Report of theCDLB International Workshop, Neurology (1996) 47:1113-24). Other LBDsinclude diffuse Lewy body disease (DLBD), Lewy body variant ofAlzheimer's disease (LBVAD), combined PD and Alzheimer's disease (AD),and multiple systems atrophy. Dementia with Lewy bodies (DLB) is a termcoined to reconcile differences in the terminology of LBDs.

Disorders with LBs continue to be a common cause for movement disordersand cognitive deterioration in the aging population (Galasko et al.,Clinical-neuropathological correlations in Alzheimer's disease andrelated dementias. Arch. Neurol. (1994) 51:888-95). Although theirincidence continues to increase, creating a serious public healthproblem, to date these disorders are neither curable nor preventable andunderstanding the causes and pathogenesis of PD is critical towardsdeveloping new treatments (Tanner et al., Epidemiology of Parkinson'sdisease and akinetic syndromes, Curr. Opin. Neurol. (2000) 13:427-30).The cause for PD is controversial and multiple factors have beenproposed to play a role, including various neurotoxins and geneticsusceptibility factors.

In recent years, new hope for understanding the pathogenesis of PD hasemerged. Specifically, several studies have shown that the synapticprotein alpha-SN plays a central role in PD pathogenesis since: (1) thisprotein accumulates in LBs (Spillantini et al., Nature (1997)388:839-40; Takeda et al., AM. J. Pathol. (1998) 152:367-72; Wakabayashiet al., Neurosci. Lett. (1997) 239:45-8), (2) mutations in the alpha-SNgene co-segregate with rare familial forms of parkinsonism (Kruger etal., Nature Gen. (1998) 18:106-8; Polymeropoulos M H, et al., Science(1997) 276:2045-7) and, (3) its overexpression in transgenic mice(Masliah et al., Science (2000) 287:1265-9) and Drosophila (Feany etal., Nature (2000) 404:394-8) mimics several pathological aspects of PD.Thus, the fact that accumulation of alpha-SN in the brain is associatedwith similar morphological and neurological alterations in species asdiverse as humans, mice, and flies suggests that this moleculecontributes to the development of PD.

An alpha-SN fragment, previously determined to be a constituent of ADamyloid plaques, was termed the non-amyloid-beta (non-Aβ) component ofAD amyloid (NAC) (Iwai A., Biochim. Biophys. Acta (2000) 1502:95-109);Masliah et al., AM J. Pathol (1996) 148:201-10; Uéda et al., Proc. Natl.Acad. Sci. USA (1993) 90:11282-6). Although the precise function of NACis not known, it may play a critical role in synaptic events, such asneural plasticity during development, and learning and degeneration ofnerve terminals under pathological conditions in LBD, AD, and otherdisorders (Hasimoto et al., Alpha-Synuclein in Lewy body disease andAlzheimer's disease, Brain Pathol (1999) 9:707-20; Masliah, et al.,(2000).

AD, PD, and dementia with Lewy bodies (DLB) are the most commonly foundneurodegenerative disorders in the elderly. Although their incidencecontinues to increase, creating a serious public health problem, to datethese disorders are neither curable nor preventable. Recentepidemiological studies have demonstrated a close clinical relationshipbetween AD and PD, as about 30% of Alzheimer's patients also have PD.Compared to the rest of the aging population, patients with AD are thusmore likely to develop concomitant PD. Furthermore, PD patients thatbecome demented usually have developed classical AD. Although eachneurodegenerative disease appears to have a predilection for specificbrain regions and cell populations, resulting in distinct pathologicalfeatures, PD, AD, DLB and LBD also share common pathological hallmarks.Patients with familial AD, Down syndrome, or sporadic AD develop LBs onthe amygdala, which are the classical neuropathological hallmarks of PD.Additionally, each disease is associated with the degeneration ofneurons, interneuronal synaptic connections and eventually cell death,the depletion of neurotransmitters, and abnormal accumulation ofmisfolded proteins, the precursors of which participate in normalcentral nervous system function. Biochemical studies have confirmed thelink between AD, PD and DLB.

The neuritic plaques that are the classic pathological hallmark of ADcontain beta-amyloid (Aβ) peptide and non-beta amyloid component (NAC)peptide. Aβ is derived from a larger precursor protein termed amyloidprecursor protein (APP). NAC is derived from a larger precursor proteintermed the non-beta amyloid component of APP, now more commonly referredto as alpha-SN. NAC comprises amino acid residues 60-87 or 61-95 ofalpha-SN. Both Aβ and NAC were first identified in amyloid plaques asproteolytic fragments of their respective full-length proteins, forwhich the full-length cDNAs were identified and cloned.

Alpha-SN is part of a large family of proteins including beta- andgamma-synuclein and synoretin. Alpha-SN is expressed in the normal stateassociated with synapses and is believed to play a role in neuralplasticity, learning and memory. Mutations in human (h) alpha-SN thatenhance the aggregation of alpha-SN have been identified (Ala30Pro andAla53Thr) and are associated with rare forms of autosomal dominant formsof PD. The mechanism by which these mutations increase the propensity ofalpha-SN to aggregate are unknown.

Despite the fact that a number of mutations can be found in APP andalpha-SN in the population, most cases of AD and PD are sporadic. Themost frequent sporadic forms of these diseases are associated with anabnormal accumulation of Aβ and alpha-SN, respectively. However, thereasons for over accumulation of these proteins is unknown. Aβ issecreted from neurons and accumulates in extracellular amyloid plaques.Additionally Aβ can be detected inside neurons. Alpha-SN accumulates inintraneuronal inclusions called LBs. Although the two proteins aretypically found together in extracellular neuritic AD plaques, they arealso occasionally found together in intracellular inclusions.

The mechanisms by which alpha-SN accumulation leads to neurodegenerationand the characteristics symptoms of PD are unclear. However, identifyingthe role of factors promoting and/or blocking alpha-SN aggregation iscritical for the understanding of LBD pathogenesis and development ofnovel treatments for its associated disorders. Research for identifyingtreatments has been directed toward searching for compounds that reducealpha-SN aggregation (Hashimoto, et al.) or testing growth factors thatwill promote the regeneration and/or survival of dopaminergic neurons,which are the cells primarily affected (Djaldetti et al., New therapiesfor Parkinson's disease, J. Neurol (2001) 248:357-62; Kirik et al.,Long-term rAAV-mediated gene transfer of GDNF in the rat Parkinson'smodel: intrastriatal but not intranigral transduction promotesfunctional regeneration in the lesioned nigrostriatal system, J.Neurosci (2000) 20:4686-4700). Recent studies in a transgenic mousemodel of AD have shown that antibodies against Aβ 1-42 facilitate andstimulate the removal of amyloid from the brain, improve AD-likepathology and resulting in improve cognitive performance (Schenk et al.,Immunization with amyloid-β attenuates Alzheimer-disease-like pathologyin PDAPP mouse, Nature (1999) 408:173-177; Morgan et al., A-beta peptidevaccination prevents memory loss in an animal model of Alzheimer'sdisease, Nature (2000) 408:982-985; Janus et al., A-beta peptideimmunization reduces behavioral impairment and plaques in a model ofAlzheimer's disease, Nature (2000) 408:979-82). In contrast to theextracellular amyloid plaques found in the brains of Alzheimer'spatients, Lewy bodies are intracellular, and antibodies do not typicallyenter the cell.

Surprisingly, given the intracellular nature of LBs in brain tissue, theinventors have succeeded in reducing the number of inclusions intransgenic mice immunized with synuclein. The present invention isdirected inter alia to treatment of PD and other diseases associatedwith LBs by administration of synuclein, fragments of synuclein,antigens that mimic synuclein or fragments thereof, or antibodies tocertain epitopes of synuclein to a patient under conditions thatgenerate a beneficial immune response in the patient. The inventors havealso surprisingly succeeded in reducing the number of inclusions intransgenic mice immunized with Aβ. The present invention is directedinter alia to treatment of PD and other diseases associated with LBs byadministration of Aβ, fragments of Aβ, antigens that mimic AP orfragments thereof, or antibodies to certain epitopes of Aβ to a patientunder conditions that generate a beneficial immune response in thepatient. The invention thus fulfills a longstanding need for therapeuticregimes for preventing or ameliorating the neuropathology and, in somepatients, the cognitive impairment associated with PD and other diseasesassociated with LBs.

This application is related to U.S. application Ser. No. 11/710,248filed Feb. 23, 2006, U.S. application Ser. No. 11/660,015 filed Feb. 9,2006, PCT Publication No. WO/2006/02058 filed Aug. 9, 2005, U.S. patentapplication Ser. No. 11/185,907, filed Jul. 19, 2005, Ser. No.10/915,214, filed Aug. 9, 2004, U.S. Pat. Nos. 6,787,523 and 6,923,964,U.S. patent application Ser. Nos. 60/423,012; 10/699,517; and10/698,099, and PCT Application No. PCT/US03/34527, each of which isincorporated by reference in its entirety for all purposes.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides methods of preventing or treatinga disease characterized by Lewy bodies or alpha-SN aggregation in thebrain. Such methods entail, inducing an immunogenic response againstalpha-SN. Such induction may be achieved by active administration of animmunogen or passive by administration of an antibody or a derivative ofan antibody to synuclein. In some methods, the patient is asymptomatic.In some methods, the patient has the disease and is asymptomatic. Insome methods the patient has a risk factor for the disease and isasymptomatic. In some methods, the disease is Parkinson's disease. Insome methods, the disease is Parkinson's disease, and the administeringthe agent improves motor characteristics of the patient. In somemethods, the disease is Parkinson's disease administering the agentprevents deterioration of motor characteristics of the patient. In somemethods, the patient is free of Alzheimer's disease.

For treatment of patients suffering from Lewy bodies or alpha-SNaggregation in the brain, one treatment regime entails administering adose of alpha-SN or an active fragment thereof to the patient to inducethe immune response. In some methods the alpha-SN or an active fragmentthereof is administered in multiple doses over a period of at least sixmonths. The alpha-SN or an active fragment thereof can be administered,for example, peripherally, intraperitoneally, orally, subcutaneously,intracranially, intramuscularly, topically, intranasally orintravenously. In some methods, the alpha-SN or an active fragmentthereof is administered with an adjuvant that enhances the immuneresponse to the alpha-SN or an active fragment thereof. In some methods,the immunogenic response comprises antibodies to alpha-SN or an activefragment thereof.

In some methods, the agent is amino acids 35-65 of alpha-SN. In somemethods, the agent comprises amino acids 130-140 of alpha-SN and hasfewer than 40 amino acids total, or fewer than 30 amino acids total. Insome methods, the agent comprises amino acids 130-136 of alpha-SN andhas fewer than 40 amino acids total, or fewer than 30 amino acids total.In some methods, the C-terminal amino acids of the agent are theC-terminal amino acid of alpha-SN. In some of the above methods, thealpha-SN or active fragment is linked to a carrier at the N-terminus ofthe alpha-SN or active fragment.

In some methods, the agent comprises amino acids 91-99 of alpha-SN andhas fewer than 40 amino acids total, or fewer than 30 amino acids total.In some methods, the agent comprises amino acids 118-126 of alpha-SN andhas fewer than 40 amino acids total, or fewer than 30 amino acids total.In some methods, the agent comprises amino acids 1-10 of alpha-SN andhas fewer than 40 amino acids total, or fewer than 30 amino acids total.In some methods, the N-terminal amino acids of the agent are theN-terminal amino acid of alpha-SN. In some of the above methods, thealpha-SN or active fragment is linked to a carrier at the C-terminus ofalpha-SN or active fragment. In some of the above methods, the alpha-SNor active fragment is linked to a carrier molecule to form a conjugate.

In some methods, the agent is administered to a patient by administeringa polynucleotide that encodes a polypeptide comprising an alpha-SNfragment.

For treatment of patients suffering from Lewy bodies or alpha-SNaggregation in the brain, one treatment regime entails administering adose of an antibody to alpha-SN or an active fragment thereof to thepatient to induce the immune response. The antibody used can be human,humanized, chimeric, or polyclonal and can be monoclonal or polyclonal.In some methods the isotype of the antibody is a human IgG1. In somemethods, the antibody is prepared from a human immunized with alpha-SNpeptide and the human can be the patient to be treated with antibody. Insome methods, the antibody binds to the outer surface of neuronal cellshaving Lewy bodies thereby dissipating the Lewy bodies. In some methods,the antibody is internalized within neuronal cells having Lewy bodiesthereby dissipating the Lewy bodies.

In some methods, the antibody is administered with a pharmaceuticalcarrier as a pharmaceutical composition. In some methods, antibody isadministered at a dosage of 0.0001 to 100 mg/kg, preferably, at least 1mg/kg body weight antibody. In some methods the antibody is administeredin multiple doses over a prolonged period, for example, at least sixmonths. In some methods antibodies can be administered as a sustainedrelease composition. The antibody can be administered, for example,peripherally, intraperitoneally, orally, subcutaneously, intracranially,intramuscularly, topically, intranasally or intravenously. In somemethods, the patient is monitored for level of administered antibody inthe blood of the patient.

In some methods, the antibody is administered by administering apolynucleotide encoding at least one antibody chain to the patient. Thepolynucleotide is expressed to produce the antibody chain in thepatient. Optionally, the polynucleotide encodes heavy and light chainsof the antibody and the polynucleotide is expressed to produce the heavyand light chains in the patient.

This invention further provides pharmaceutical compositions comprisingany of the antibodies to alpha-SN described in this application and apharmaceutically acceptable carrier.

In another aspect, the invention provides methods of preventing ortreating a disease characterized by Lewy bodies or alpha-SN aggregationin the brain comprising administering an agent that induces animmunogenic response against alpha-SN, and further comprisingadministering of a second agent that induces an immunogenic responseagainst Aβ to the patient. In some methods, the agent is Aβ or an activefragment thereof. In some methods, the agent is an antibody to Aβ.

In another aspect, the invention provides methods of preventing ortreating a disease characterized by Lewy bodies or alpha-SN aggregationin the brain comprising administering an agent that induces animmunogenic response against Aβ to a patient. In some methods, the agentis Aβ or an active fragment thereof. In some methods, the agent is anantibody to Aβ. In some methods the disease is Parkinson's disease. Insome methods, the patient is free of Alzheimer's disease and has no riskfactors thereof. In some methods, further comprise monitoring a sign orsymptom of Parkinson's disease in the patient. In some methods, thedisease is Parkinson's disease and administering the agent results inimprovement in a sign or symptom of Parkinson's disease.

This invention further provides pharmaceutical compositions comprisingan agent effective to induce an immunogenic response against a componentof a Lewy body in a patient, such as described above, and apharmaceutically acceptable adjuvant. In some compounds, the agent isalpha-SN or an active fragment, for example, NAC, or any of thefragments described in the application. The invention also providespharmaceutical compositions comprising an antibody specific for acomponent of a Lewy body.

This invention also provides pharmaceutical compositions comprising anagent effective to elicit an immune response against a synuclein-NACcomponent of an amyloid plaque in a patient. In some compounds, theagent is alpha-SN or an active fragment, for example, NAC, or any of thefragments of alpha synuclein described in the application, and,optionally, an adjuvant. In other compounds, the agent is an antibody orfragment thereof that specifically binds alpha-SN or a fragment thereof,and, optionally, a pharmaceutical carrier.

In another aspect, the invention provides for methods of screening anantibody for activity in preventing or treating a disease associatedwith Lewy bodies. Such methods entail, contacting a neuronal cellexpressing synuclein with the antibody. Then one determines whether thecontacting reduces synuclein deposits in the cells compared with acontrol cells not contacted with the antibody.

In another aspect, the invention provides for methods of screening anantibody for activity in treating or preventing a Lewy body disease inthe brain of a patient. Such methods entail contacting the antibody witha polypeptide comprising at least five contiguous amino acids ofalpha-SN. Then one determines whether the antibody specifically binds tothe polypeptide, specific binding providing an indication that theantibody has activity in treating the disease.

The invention further provides methods of effecting prophylaxis ortreating a disease characterized by Lewy bodies or alpha-synucleinaggregation in the brain. The method comprises administering to apatient having or at risk of the disease a polypeptide comprising animmunogenic fragment of alpha-synuclein effective to induce animmunogenic response comprising antibodies that specifically bind to anepitope within residues 70-140 of human alpha-synuclein, residues beingnumbered according to SEQ ID NO:1, thereby effecting prophylaxis ortreatment of the disease.

Optionally, the immunogenic fragment of alpha-synuclein is free ofresidues 1-69 of alpha synuclein, residues being numbered according toSEQ ID NO:1. Optionally, the immunogenic response comprises antibodiesthat specifically binds to human alpha synuclein within an epitopeselected from the group consisting of SN83-101, SN107-125, SN110-128 andSN124-140, residues being numbered according to SEQ ID NO:1. Optionally,the immunogenic response is free of antibodies that specifically bind toresidues of human alpha synuclein outside the selected epitope.Optionally, the immunogenic fragment has from 5-20, 5-25 or 5-30contiguous amino acids from between positions 70-140 of alpha synuclein,residues being numbered according to SEQ ID NO:1. Optionally, theimmunogenic fragment has from 5-20 contiguous amino acids from betweenpositions 120-140 of alpha synuclein, residues being numbered accordingto SEQ ID NO:1. Optionally, the immunogenic fragment comprises a segmentof human alpha synuclein selected from the group consisting of SN87-97,SN111-121, SN114-124 and SN128-136, and contains no more than 40, or nomore than 30, contiguous residues in total of alpha synuclein, residuesbeing numbered according to SEQ ID NO:1. Optionally, the immunogenicfragment comprises a segment of human alpha synuclein selected from thegroup consisting of SN 124-134, SN 91-99 and SN 118-126 and contains nomore than 40, or no more than 30, contiguous residues in total of alphasynuclein, residues being numbered according to SEQ ID NO:1 Optionally,the immunogenic fragment comprises SN125-140 and contains no more than40, or no more than 30, contiguous residues in total of alpha synuclein,residues being numbered according to SEQ ID NO:1. Optionally, theimmunogenic fragment comprises SN130-140 and contains no more than 40,or no more than 30, contiguous residues in total of alpha synuclein,residues being numbered according to SEQ ID NO:1. Optionally, theimmunogenic fragment comprises SN83-140, residues being numberedccording to SEQ ID NO:1.

Optionally, the immunogenic fragment is selected from a group consistingof SN124-140, SN125-140, SN126-140, SN127-140, SN128-140, SN 129-140,SN130-140, SN131-140, SN132-140, SN133-140, SN134-140, SN135-140,SN136-140, SN137-140, SN124-139, SN125-139, SN126-139, SN127-139,SN128-139, SN124-139, SN125-139, SN126-139, SN127-139, SN128-139, SN129-139, SN130-139, SN131-139, SN132-139, SN133-139, SN134-139,SN135-139, SN136-139, SN137-139, SN124-138, SN124-138, SN125-138,SN126-138, SN127-138, SN128-138, SN 129-138, SN130-138, SN131-138,SN132-138, SN133-138, SN134-138, SN135-138, SN136-138, SN124-137,SN125-137, SN126-137, SN127-137, SN128-137, SN 129-137, SN130-137,SN131-137, SN132-137, SN133-137, SN134-137, SN135-137, SN124-136,SN125-136, SN126-136, SN127-136, SN128-136, SN 129-136, SN130-136,SN131-136, SN132-136, SN133-136, and SN134-136, residues being numberedaccording to SEQ ID NO:1.

Optionally, the immunogenic response comprises antibodies thatspecifically bind to human alpha synuclein within an epitope selectedfrom the group consisting of SN1-20, SN2-21, SN2-23 and SN1-40, residuesbeing numbered according to SEQ ID NO:1. Optionally, the immunogenicresponse is free of antibodies that specifically bind to residues ofhuman alpha synuclein within the region SN25-69, SN25-140, SN40-69,SN40-140, or SN70-140. Optionally, the immunogenic fragment has from5-20, 5-25 or 5-30 contiguous amino acids from between positions 1-40 ofalpha synuclein, residues being numbered according to SEQ ID NO:1.

Optionally, the immunogenic fragment has from 5-20 contiguous aminoacids from between positions 1 and 20 and from 5-20 contiguous aminoacids from between positions 120 and 140 of alpha synuclein, residuesbeing numbered according to SEQ ID NO:1. Optionally the immunogenicfragment contains no more than 40, or no more than 30, contiguousresidues in total of alpha synuclein, residues being numbered accordingto SEQ ID NO:1.

Optionally, the immunogenic response comprises antibodies thatspecifically bind to human alpha synuclein within an epitope withinresidues 1-20 of human alpha-synuclein and within an epitope withinresidues 70-140 of human alpha-synuclein. Optionally, the immunogenicresponse comprises antibodies that specifically binds to human alphasynuclein within an epitope within residues 1-20 of humanalpha-synuclein and within an epitope within residues 120-140 of humanalpha-synuclein. Optionally, the immunogenic response does not compriseantibodies that specifically bind to human alpha synuclein within anepitope within residues 41 and 119 of human alpha-synuclein.

Optionally, the immunogenic fragment is linked to a carrier to form aconjugate. Optionally, the polypeptide comprises the immunogenicfragment fused to the carrier. Optionally, the immunogenic fragment islinked to the carrier molecule at the C-terminus of the alpha-synucleinfragment. Optionally, multiple copies of the fragment are interlinkedwith multiple copies of the carrier. Optionally, the immunogenicfragment is administered with an adjuvant. Optionally, the administeringstep effects at least partial clearance of Lewy Bodies. Optionally, theadministering step disaggregates Lewy Bodies. Optionally, theadministering step reduces levels of alpha synuclein oligomers insynapses. Optionally, the administering step clears synuclein byactivation of a lysosomal pathway.

Optionally the immunogenic response is induced by administration of asingle polypeptide or fusion protein. Optionally the immunogenicresponse is induced by administration of more than one polypeptide(e.g., two polypeptides). Optionally the immunogenic response is inducedby administration of a first polypeptide comprising a first immunogenicfragment of alpha-synuclein effective to induce an immunogenic responsecomprising antibodies that specifically bind to an epitope withinresidues 1-20 of human alpha-synuclein, and administering a polypeptidecomprising a second immunogenic fragment of alpha-synuclein effective toinduce an immunogenic response comprising antibodies that specificallybind to an epitope within residues 70-140, and preferably residues120-140, of human alpha-synuclein.

Optionally, the immunogenic response is induced by administration of twoor more polypeptides in combination. Optionally the two or morepolypeptides are co-administered and/or co-formulated.

In another aspect, the invention provides a composition comprising afirst polypeptide comprising a first immunogenic fragment ofalpha-synuclein and a second polypeptide comprising a second immunogenicfragment of alpha-synuclein, where the first immunogenic fragment iseffective to induce an immunogenic response comprising antibodies thatspecifically bind to an epitope within residues 1-20 of humanalpha-synuclein and the second immunogenic fragment of alpha-synucleinis effective to induce an immunogenic response comprising antibodiesthat specifically bind to an epitope within residues 120-140 of humanalpha-synuclein. Optionally the composition is free of an immunogenicfragment of alpha-synuclein comprising residues 25-69 ofalpha-synuclein. The first and second immunogenic fragments can bephysically linked (e.g., as a conjugate or fusion protein). The firstand second immunogenic fragments can be coformulated.

The invention further provides methods of effecting prophylaxis ortreating a disease characterized by Lewy bodies or alpha-synucleinaggregation in the brain. In one embodiment the methods compriseadministering to a patient having or at risk of the disease an effectiveregime of an antibody that specifically binds to an epitope withinresidues 70-140 of human alpha-synuclein, residues being numberedaccording to SEQ ID NO:1. When the antibody specifically binds to anepitope within residues 70-140 of human alpha-synuclein, residues beingnumbered according to SEQ ID NO:1, optionally, the antibody specificallybinds to an epitope within residues 83-140 of human alpha synuclein,residues being numbered according to SEQ ID NO:1. Optionally, theantibody specifically binds to an epitope within residues 120-140 ofhuman alpha synuclein. Optionally, the antibody specifically bindswithin an epitope within a segment of human alpha synuclein selectedfrom the group consisting of SN83-101, SN107-125, SN110-128, SN 118-126,SN 91-99, SN 124-134 and SN124-140, residues being numbered according toSEQ ID NO:1.

In another embodiment the methods comprise administering to a patienthaving or at risk of the disease an effective regime of an antibody thatspecifically binds to an epitope within residues 1-40 of humanalpha-synuclein, residues being numbered according to SEQ ID NO:1. Whenthe antibody specifically binds to an epitope within residues 1-40 ofhuman alpha-synuclein, residues being numbered according to SEQ ID NO:1,optionally the antibody specifically binds to an epitope within residues1-20, or within residues 1-10, residues being numbered according to SEQID NO:1.

In still another embodiment the methods comprise administering to apatient having or at risk of the disease an effective regime of a firstantibody that specifically binds to an epitope within residues 1-40 ofhuman alpha-synuclein and a second antibody that specifically binds toan epitope within residues 70-140 of human alpha-synuclein, residuesbeing numbered according to SEQ ID NO:1.

When a first antibody that specifically binds to an epitope withinresidues 1-40 of human alpha-synuclein and a second antibody thatspecifically binds to an epitope within residues 70-140 of humanalpha-synuclein, residues being numbered according to SEQ ID NO:1,optionally the second antibody specifically binds to an epitope withinresidues 120-140 of human alpha-synuclein. Optionally the first antibodyand second antibody are administered simultaneously. Optionally thefirst antibody and second antibody are administered in the same courseof treatment.

Optionally, the antibody is a monoclonal antibody. Optionally, theantibody is a polyclonal population of antibodies lacking specificbinding to residues of alpha synuclein outside the epitope. Optionally,the antibody is a humanized antibody. Optionally, the antibody is humanantibody. Optionally, the antibody is an antibody of human IgG1 isotype.Optionally, the antibody is administered with a pharmaceutical carrieras a pharmaceutical composition. Optionally, the antibody isadministered at a dosage of 0.0001 to 100 mg/kg, preferably, at least 1mg/kg body weight antibody. Optionally, the antibody is administered inmultiple dosages over at least six months. Optionally, the antibody isadministered as a sustained release composition. Optionally, theantibody is administered intraperitoneally, orally, subcutaneously,intracranially, intramuscularly, topically, intranasally orintravenously. Optionally, the antibody is internalized within neuronalcells having Lewy bodies thereby dissipating the Lewy bodies.Optionally, the antibody binds to the outer surface of neuronal cellshaving Lewy bodies thereby dissipating the Lewy bodies. Optionally, theantibody binds to alpha synuclein on the outer surface of neuronal cellspromoting crosslinking of the alpha synuclein. wherein the administeringstep disaggregates Lewy bodies. Optionally, the administering stepreduces levels of human alpha synuclein oligomers in synapses.Optionally, the administering step clears human alpha synuclein byactivation of a lysosomal pathway. In some methods, the disease isParkinson's disease. Optionally, the antibody specifically binds todenatured human alpha-synuclein as determined by immunoblot. Optionally,the antibody specifically binds to denatured human alpha-synuclein withan affinity of at least 10⁹ M⁻¹. Optionally, the antibody specificallybinds to synapses as determined by immunocytochemistry.

The invention also provides a composition for prophylaxis or treatmentof a disease characterized by Lewy bodies or alpha-synuclein aggregationin the brain, comprising a first monoclonal antibody that specificallybinds to an epitope within residues 1-20 of human alpha-synuclein and asecond monoclonal antibody that specifically binds to an epitope withinresidues 70-140 (and preferably residues 120-140) of humanalpha-synuclein, residues being numbered according to SEQ ID NO:1.

The invention also provides a kit for prophylaxis or treatment of adisease characterized by Lewy bodies or alpha-synuclein aggregation inthe brain, comprising a first container comprising an antibody thatspecifically binds to an epitope within residues 1-20 of humanalpha-synuclein and a second container comprising an antibody that thatspecifically binds to an epitope within residues 70-140 (and preferablyresidues 120-140) of human alpha-synuclein, residues being numberedaccording to SEQ ID NO:1.

The invention further provides methods of effecting prophylaxis ortreating a disease characterized by Lewy bodies or alpha-synucleinaggregation in the brain. The methods comprise administering to apatient suffering from or at risk of the disease an effective regime ofan agent that induces an immunogenic response comprising antibodies thatspecifically bind to an epitope within residues 70-140 of human alphasynuclein, residues being numbered according to SEQ ID NO:1, therebyeffecting prophylaxis or treating the disease. Optionally, theimmunogenic response is free of antibodies that specifically bind to anepitope within residues 1-69 of human alpha synuclein, residues beingnumbered according to SEQ ID NO:1. Optionally, the immunogenic responsecomprises antibodies that specifically bind within a segment of humanalpha synuclein selected from the group consisting of SN83-101,SN107-125, SN110-128, SN 118-126, SN 91-99, SN 124-134 and SN124-140,residues being numbered according to SEQ ID NO:1.

The invention further provides methods of screening for an agent hasactivity useful in treating a disease characterized by Lewy Bodies. Themethods comprise contacting the agent with a transgenic nonhuman animaldisposed to develop a characteristic of a Lewy Body disease with theagent; and determining whether the agent affects the extent or rate ofdevelopment of the characteristic relative to a control transgenicnonhuman animal. The agent is (i) an fragment of alpha synuclein thatinduces antibodies that specifically bind to at least one epitope withinresidues 70-140 of human alpha synuclein or (ii) an antibody thatspecifically binds to an epitope with residues 70-140 of human alphasynuclein, residues being numbered according to SEQ ID NO:1.

Optionally, the transgenic nonhuman animal comprises a transgeneexpressing human alpha-synuclein. Optionally, the method furthercomprises screening a plurality of test antibodies for binding todenatured human alpha synuclein, and selecting the highest bindingantibody as the agent. Optionally, the method further comprisesscreening a plurality of test antibodies for binding to deposits ofsynuclein in a tissue section by immunocytochemistry, and selecting thehighest binding antibody as the agent.

The invention further provides a method of humanizing a monoclonalantibody selected from 8A5, 6H7, 9E4, 1H7, and 11A5 comprising:determining the amino acid sequence of CDR regions of the monoclonalantibody; selecting an acceptor antibody; and producing a humanizedantibody comprising the CDRs from the monoclonal antibody and variableregion frameworks from the acceptor antibody.

The invention further provides a method of producing a chimeric form ofa monoclonal antibody selected from 8A5, 6H7, 9E4, 1H7, and 11A5,comprising: determining the amino acid sequence of the light and heavychain variable regions of the monoclonal antibody; selecting heavy andlight chain constant region; producing a chimeric antibody comprising alight chain comprising the light chain variable region fused to thelight chain constant region, and a heavy chain comprising the heavychain variable region fused to the heavy chain constant region.

The invention provides methods of effecting prophylaxis or treating adisease characterized by Lewy bodies or alpha-synuclein aggregation inthe brain, the method comprising administering to a patient having or atrisk of the disease an effective regime of an antibody that specificallybinds to an epitope within residues 110-130 of human alpha-synuclein,residues being numbered according to SEQ ID NO:1. Optionally, theantibody binds to an epitope within residues 118-126 of humanalpha-synuclein. Optionally the disease is Parkinson's disease.Optionally, the antibody is a monoclonal antibody. Optionally, theantibody is a chimeric antibody, a human antibody, or a humanizedantibody. Optionally, the antibody competes with mouse monoclonalantibody 9E4 (ATCC accession number PTA-8221) for binding to humanalpha-synuclein. Optionally, the antibody is monoclonal antibody 9E4(ATCC accession number PTA-8221) or humanized or chimeric 9E4.Optionally, the antibody is an antibody of human IgG1 isotype.Optionally, the antibody is administered with a pharmaceutical carrieras a pharmaceutical composition. Optionally, the antibody isadministered at a dosage of 0.0001 to 100 mg antibody/kg body weight.Optionally, the antibody is administered in multiple dosages over atleast six months. Optionally, the antibody is administeredintraperitoneally, orally, subcutaneously, intracranially,intramuscularly, topically, intranasally or intravenously. Optionally,the antibody is administered by a peripheral route. Optionally, theantibody is administered at a dose of 1-10 mg/kg.

The invention further provides a monoclonal antibody 9E4 (ATCC accessionnumber PTA-8221) or humanized or chimeric 9E4.

The invention further provides a method of humanizing monoclonalantibody 9E4 (ATCC accession number PTA-8221) comprising: determiningthe amino acid sequence of CDR regions of the monoclonal antibody;selecting an acceptor antibody; and producing a humanized antibodycomprising the CDRs from the monoclonal antibody and variable regionframeworks from the acceptor antibody.

The invention further provides a method of producing a chimeric form ofmonoclonal antibody 9E4 (ATCC accession number PTA-8221), comprising:determining the amino acid sequence of the light and heavy chainvariable regions of the monoclonal antibody; selecting heavy and lightchain constant region; and producing a chimeric antibody comprising alight chain comprising the light chain variable region fused to thelight chain constant region, and a heavy chain comprising the heavychain variable region fused to the heavy chain constant region.

The invention further provides an anti-synuclein monoclonal antibodyproduced by hybridoma JH17.9E4.3.37.1.14.2 (ATCC accession numberPTA-8221), hybridoma JH17.1H7.4.24.34 (ATCC accession number PTA-8220),or hybridoma JH22.11A5.6.29.70.54.16.14 (ATCC accession numberPTA-8222).

The invention further provides a chimeric or humanized antibody of amonoclonal antibody produced by hybridoma JH17.1H7.4.24.34 (ATCCaccession number PTA-8220), or hybridoma JH22.11A5.6.29.70.54.16.14(ATCC accession number PTA-8222).

The invention further provides a method of humanizing a monoclonalantibody produced by hybridoma JH17.1H7.4.24.34 (ATCC accession numberPTA-8220), or hybridoma JH22.11A5.6.29.70.54.16.14 (ATCC accessionnumber PTA-8222) comprising: determining the amino acid sequence of CDRregions of the monoclonal antibody; selecting an acceptor antibody; andproducing a humanized antibody comprising the CDRs from the monoclonalantibody and variable region frameworks from the acceptor antibody.

The invention further provides a method of producing a chimeric form ofa monoclonal antibody produced by hybridoma JH17.1H7.4.24.34 (ATCCaccession number PTA-8220), or hybridoma JH22.11A5.6.29.70.54.16.14(ATCC accession number PTA-8222), comprising: determining the amino acidsequence of the light and heavy chain variable regions of the monoclonalantibody; selecting heavy and light chain constant region; producing achimeric antibody comprising a light chain comprising the light chainvariable region fused to the light chain constant region, and a heavychain comprising the heavy chain variable region fused to the heavychain constant region.

The invention further provides a cell of hybridoma JH17.9E4.3.37.1.14.2(ATCC accession number PTA-8221), hybridoma JH17.1H7.4.24.34 (ATCCaccession number PTA-8220), or hybridoma JH22.11A5.6.29.70.54.16.14(ATCC accession number PTA-8222).

The invention further provides a use of an antibody that specificallybinds to an epitope within residues 110-130 of human alpha-synuclein,residues being numbered according to SEQ ID NO:1 in the manufacture of amedicament to treat a disease characterized by Lewy bodies or alphasynuclein aggregation whereby the antibody is administered by aperipheral route, and reduces intracellular Lewby bodies or alphasynuclein aggregation by a lysosomal pathway. Optionally, the antibodyenters a cell containing a Lewy body or alpha synuclein aggregation byendocytosis. Optionally, the antibody binds to membrane bound alphasynuclein from the exterior of the cell and a complex of antibody andalpha synuclein are endocytosed into the cell and combine with alysosome within the cell. Optionally, the antibody and alpha synculeinare separately endocytosed and combined with a lysosome within the cell.

The invention further provides a method of effecting prophylaxis ortreating a disease characterized by Lewy bodies or alpha-synucleinaggregation in the brain, the method comprising administering to apatient having or at risk of the disease an effective regime of an agentthat induces an antibody that specifically binds to an epitope withinresidues 110-130 of human alpha-synuclein, residues being numberedaccording to SEQ ID NO:1. Optionally, the agent is a fragment ofalpha-synuclein. Optionally, the fragment has no more than 30 contiguousresidues total of alpha-synuclein. Optionally, the fragment induces anantibody to an eptiope within residues 118-126 of alpha-synuclein.Optionally, the fragment comprises residues 118-126 of alpha synuclein.Optionally, the fragment is linked to a carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of alpha-SN (SEQ ID: 1) inalignment with two NAC amino acid sequences, SEQ ID NO: 2 and SEQ ID NO:3, respectively.

FIG. 2 shows immunohistostained brain sections from nontransgenic mice(panels A, E, and I), alpha-SN transgenic mice immunized with adjuvantalone (panels B, F, J), and alpha-SN transgenic mice immunized withalpha-SN which developed low titers (panels C, G, and K) and high titers(panels D, H, and I) of antibodies to alpha-SN. Sections were subjectedto staining with an anti-alpha-synulcein antibody to detect synucleinlevels (panels A-D), an anti-IgG antibody to determine total IgG levelspresent in the section (panels E-H), and for Glial Fibrillary AcidicProtein (GFAP), a marker of astroglial cells.

FIGS. 3A-D show the effects of anti-mSYN polyclonal antibody onsynuclein aggregation in transfected GT1-7 cells as seen by lightmicroscopy.

FIG. 4 is a Western blot of synuclein levels in the cytoplasm (C) andmembranes (P) of GT1-7 α-syn cells treated with preimmune sera and with67-10 antibody at a concentration of (1:50) for 48 hours prior toanalysis.

FIG. 5 shows the results of studies of the effect of Aβ1-42 immunizationamyloid deposition in the brains of nontransgenic, SYN, APP and SYN/APPtransgenic mice. The detectable amyloid levels seen in APP and SYN/APPmice are reduced by Aβ1-42 immunization.

FIG. 6 shows the results of studies of the effect of Aβ1-42 immunizationupon synuclein inclusion formation in the brains of nontransgenic, SYN,APP and SYN/APP transgenic mice. Synulcein inclusions detected in SYNand SYN/APP mice are reduced by Aβ1-42 immunization.

FIG. 7 shows direct and indirect mechanisms by which antibodies blockalpha-SN aggregation.

FIG. 8 shows antibody epitope mapping. Antibodies from mice displayinghigh titers and high affinity anti-human α-synuclein antibodies weremapped using an ELISA technique. In most anti-sera samples fromvaccinated mice, epitopes recognized were within the C-terminal regionof human α-synuclein. In the sera from CFA treated controls, no epitopeswere detected.

FIG. 9 shows image analysis of the levels of human α-synucleinimmunoreactivity and other markers of neurodegeneration. (A) Mean numberof ha-synuclein positive inclusions in the temporal cortex. Vaccinationwith human α-synuclein resulted in a significant decrease in the numberof inclusions compared to controls. This effect was more pronounced inmice from group II as opposed to group I. (B) Percent area of theneuropil occupied by synaptophysin-immunoreactive terminals in thefrontal cortex. In transgenic (tg) mice treated with CFA alone, thenumber of synaptophysin immunolabeled terminals decreased by 20%,whereas the levels of synaptophysin immunoreactivity per synapse wasunchanged. (C) Levels of CD45 immunoreactivity (microglial marker) inthe temporal cortex were slightly higher in the brains of humanα-synuclein vaccinated mice. (D) Percent area of the neuropil ofoccupied by human α-synuclein immunoreactive terminals in the temporalcortex. In tg mice vaccinated with human α-synuclein, there was adecrease in the accumulation of α-synuclein insynaptophysin-immunoreactive terminals. *=significant differencecompared to human α-synuclein tg mice treated with CFA alone (p<0.05,student's T test).

FIG. 10 shows Western blot analysis of the levels of human α-synucleinand synaptophysin immunoreactivity in vaccinated animals. Compared tobrains of tg mice treated with CFA alone (lanes 1-3), in ha-synucleinvaccinated tg mice (lanes 4-6), levels of both human α-synucleinoligomers and monomers were decreased (upper panel), whereas levels ofsynaptophysin immunoreactivity increased in the latter group (lowerpanel).

FIG. 11 shows analysis of intraneuronal α-synuclein aggregates afterintracerebral injection of anti-α-synuclein antibodies. The C-terminalantibody 8A5 and the N-terminal antibody 6H7 had a clearing effect.IgG1, IgG2a, and IgG2b were isotype controls. Horizontal bars representthe median.

FIG. 12 shows sections of the contralateral side (left panel; roundbrown dots inside section are α-synuclein aggregates) and ipsilateralside (right panel) of a mouse injected with monoclonal antibody 8A5.Immunostaining was performed with a polyclonal antibody for α-synuclein.Intraneuronal α-synuclein aggregates in the contralateral side arecircled.

FIG. 13 shows clearance of intraneuronal α-synuclein aggregates in theneocortex of transgenic mice overexpressing human α-synuclein byanti-α-synuclein monoclonal antibodies 8A5, 9E4, and 6H7.

FIG. 14 shows aggregation of α-synuclein in the membrane andlocalization of α-synuclein to lysozomes in human α-synuclein mice aftera single intraveneous injection with 9E4-FITC.

FIG. 15 shows reduced levels of α-syn oligomers and insoluble forms ofα-syn were found in the brains of tg mice injected intraperitoneallywith 9E4 at a dosage of 1 mg/kg for six months.

FIG. 16 shows levels of beclin-1, LC3 cleavage and Atg5 were increasedand co-localized with the α-syn aggregates in the neurons of immunizedtg mice.

FIG. 17 shows how an antibody against an epitope at or near thec-terminus of α-syn traffics into the CNS, recognizes aggregates inaffected neurons and triggers clearance via a lysosomal pathway, such asautophagy.

DEFINITIONS

The term “substantial identity” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 65 percent sequence identity, preferably atleast 80 or 90 percent sequence identity, more preferably at least 95percent sequence identity or more (e.g., 99 percent sequence identity orhigher). Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra). One example of algorithm that is suitable fordetermining percent sequence identity and sequence similarity is theBLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(NCBI) website. Typically, default program parameters can be used toperform the sequence comparison, although customized parameters can alsobe used. For amino acid sequences, the BLASTP program uses as defaults aword length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915(1989)).

For purposes of classifying amino acids substitutions as conservative ornon-conservative, amino acids are grouped as follows: Group I(hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic sidechains): asp, glu; Group IV (basic side chains): asn, gln, his, lys,arg; Group V (residues influencing chain orientation): gly, pro; andGroup VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained.

The phrase that a molecule “specifically binds” to a target refers to abinding reaction which is determinative of the presence of the moleculein the presence of a heterogeneous population of other biologics. Thus,under designated immunoassay conditions, a specified molecule bindspreferentially to a particular target and does not bind in a significantamount to other biologics present in the sample. Specific binding of anantibody to a target under such conditions requires the antibody beselected for its specificity to the target. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity. Specific binding betweentwo entities means an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹ M⁻¹, or10¹⁰ M⁻¹. Affinities greater than 10⁸ M⁻¹ are preferred.

The term “antibody” or “immunoglobulin” is used to include intactantibodies and binding fragments thereof. Typically, fragments competewith the intact antibody from which they were derived for specificbinding to an antigen. Fragments include separate heavy chains, lightchains, Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes one or moreimmunoglobulin chains that are chemically conjugated to, or expressedas, fusion proteins with other proteins. The term “antibody” alsoincludes bispecific antibody. A bispecific or bifunctional antibody isan artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553(1992). The term “antibody” also includes single-chain antibodies inwhich heavy and light chain variable domains are linked through aspacer.

APP⁶⁹⁵, APP⁷⁵¹, and APP⁷⁷⁰ refer, respectively, to the 695, 751, and 770amino acid residue long polypeptides encoded by the human APP gene. SeeKang et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525(1988); and Kitaguchi et al., Nature 331, 530 (1988). Amino acids withinthe human amyloid precursor protein (APP) are assigned numbers accordingto the sequence of the APP770 isoform. Terms such as Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43 refer to an Aβ peptide containing amino acid residues1-39, 1-40, 1-41, 1-42 and 1-43.

An “antigen” is an entity to which an antibody specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which B and/or T cells respond. B-cell epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen. T-cells recognize continuous epitopes ofabout nine amino acids for CD8 cells or about 13-15 amino acids for CD4cells. T cells that recognize the epitope can be identified by in vitroassays that measure antigen-dependent proliferation, as determined by³H-thymidine incorporation by primed T cells in response to an epitope(Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependentkilling (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156,3901-3910) or by cytokine secretion.

The term “immunological” or “immune” response is the development of abeneficial humoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst an amyloid peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. A cellular immune response is elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules to activate antigen-specific CD4⁺ T helper cells and/or CD8⁺cytotoxic T cells. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic Tlymphocyte) assays (see Burke, supra; Tigges, supra). The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating antibodies and T-cells from an immunized syngeneic animal andmeasuring protective or therapeutic effect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

The term “all-D” refers to peptides having 75%, 80%, 85%, 90%, 95%, and100% D-configuration amino acids.

The term “naked polynucleotide” refers to a polynucleotide not complexedwith colloidal materials. Naked polynucleotides are sometimes cloned ina plasmid vector.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments the immune response to the antigen,but when administered alone does not generate an immune response to theantigen. Adjuvants can augment an immune response by several mechanismsincluding lymphocyte recruitment, stimulation of B and/or T cells, andstimulation of macrophages.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as alpha-SN. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, Antibodies, A Laboratory Manual,Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabelled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50% or 75%.

The term “symptom” or “clinical symptom” refers to a subjective evidenceof a disease, such as altered gait, as perceived by the patient. A“sign” refers to objective evidence of a disease as observed by aphysician.

The phrase “in combination,” when referring to administration of two ormore anti-human alpha-synuclein antibodies (i.e., each recognizing adifferent epitope) or administration of two or more polypeptides orimmunogens that induce an antibody response against humanalpha-synuclein, includes simultaneous administration and administrationin the same course of treatment. Simultaneous administration of agentsencompasses administration of the agents as a fusion protein orconjugate (e.g., physically linked to each other), a co-formulation(e.g., in which the agents are combined or compounded into a dosageform, e.g., a sustained release or depot formulation), administration asseparate compositions within a few minutes or two hours of each other(co-administration), or administration as separate compositions on thesame day. Administration in the same course of treatment means bothagents are administered to a patient for treatment or prophylaxis of thesame condition. Each agent can be administered once or multiple times.For example, one agent might be administered first and the second agentadministered the following day or following week. Similarly, the twoagents might each be administered more than once, e.g., on sequentialdays, alternate days, alternate weeks, or according to other schedules(for example, such that the benefit to the patient is expected to exceedthat of administration of either agent alone).

A fragment designated in the form SNx-y means a fragment of alphasynuclein that begins at amino acid X and ends at amino acid Y. Such afragment can be linked to a heterologous polypeptide but not to otheramino acids of human alpha synuclein such that the fragment beginsbefore X or ends after Y.

Residues in alpha-synuclein or a fragment thereof are numbered accordingto SEQ ID NO:1 when alpha-synuclein or the fragment is maximally alignedwith SEQ ID NO:1 as described above using default parameters.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises alpha-SN peptide encompasses both an isolatedalpha-SN peptide and alpha-SN peptide as a component of a largerpolypeptide sequence.

DETAILED DESCRIPTION OF THE INVENTION I. General

The invention provides methods of preventing or treating severaldiseases and conditions characterized by presence of deposits ofalpha-SN peptide aggregated to an insoluble mass in the brain of apatient, in the form of Lewy bodies. Such diseases are collectivelyreferred to as Lewy Body diseases (LBD) and include Parkinson's disease(PD). Such diseases are characterized by aggregates of alpha-SN thathave a β-pleated sheet structure and stain with thioflavin-S andCongo-red (see Hasimoto, Mid). The invention provides methods ofpreventing or treating such diseases using an agent that can generate animmunogenic response to alpha-SN. The immunogenic response acts toprevent formation of, or clear, synuclein deposits within cells in thebrain. Although an understanding of mechanism is not essential forpractice of the invention, the immunogenic response can induce clearingas a result of antibodies to synuclein that are internalized withincells alone or with alpha synuclein. The results presented in theExamples show that antibodies to alpha synuclein administeredperipherally cross the blood brain barrier, and are internalized, eitheralone or with alpha synuclein, within cells containing alpha synucleindeposits. Internalized antibodies can promote degradation of alphasynuclein via activation of lysosomal pathways. Internalized antibodieswith affinity for alpha synuclein in denatured form can also stabilizethe molecule in unaggregated form. Alternatively or additionally,antibodies can interfere with aggregation of synuclein on the cellexterior surface. For example, antibodies to alpha-synuclein mayrecognized and crosslink abnormally conformed proteins in the neuronalcells surface. In some methods, the clearing response can be effected atleast in part by Fc receptor mediated phagocytosis. Immunization withsynuclein can reduce synuclein accumulation at synapses and neuronalcell bodies in the brain. Although an understanding of mechanism is notessential for practice of the invention, this result can be explained byantibodies to synuclein being taken up by neuronal cells (e.g., bysynaptic vesicles).

Optionally, agents generating an immunogenic response against alpha-SNcan be used in combination with agents that generate an immunogenicresponse to Aβ. The immunogenic response is useful in clearing depositsof Aβ in individuals having such deposits (e.g., individuals having bothAlzheimer's and Parkinson's disease); however, the immunogenic responsealso has activity in clearing synuclein deposits. Thus, the presentinvention uses such agents alone, or in combination with agentsgenerating an immunogenic response to alpha-SN in individuals with LBDbut who are not suffering or at risk of developing Alzheimer's disease.

The invention further provides pharmaceutical compositions and methodsfor preventing and treating amyloidogenic disease. It has been shownthat alpha- and beta synuclein are involved in nucleation of amyloiddeposits in certain amyloid diseases, particularly Alzheimer's disease.(Clayton, D. F., et al., TINS 21(6): 249-255, 1998). More specifically,a fragment of the NAC domain of alpha- and beta-synuclein (residues61-95) has been isolated from amyloid plaques in Alzheimer's patients;in fact this fragment comprises about 10% of the plaque that remainsinsoluble after solubilization with sodium dodecyl sulfate (SDS).(George, J. M., et al. Neurosci. News 1: 12-17, 1995). Further, both thefull length alpha-SN and the NAC fragment thereof have been reported toaccelerate the aggregation of β-amyloid peptide into insoluble amyloidin vitro. (Clayton, supra). The NAC component of amyloid plaques servesas a target for immunologically-based treatments of the presentinvention, as detailed below. According to one aspect, the inventionincludes pharmaceutical compositions comprising agents effective toelicit an immune response against a synuclein-NAC component of anamyloid plaque in a patient. Such compositions can be effective inpreventing, retarding, or reducing plaque deposition in amyloid disease.

II. Agents Generating an Immunogenic Response Against Alpha Synuclein

An immunogenic response can be active, as when an immunogen isadministered to induce antibodies reactive with alpha-SN in a patient,or passive, as when an antibody is administered that itself binds toalpha-SN in a patient.

1. Agents Inducing Active Immune Response

Therapeutic agents induce an immunogenic response specifically directedto certain epitopes within the alpha-SN peptide. Preferred agents arethe alpha-SN peptide itself and fragments thereof. U.S. patentpublication US20060259986A1 and PCT patent publication WO 05/013889,both of which are incorporated herein by reference for all purposes,disclose novel alpha-synuclein fragments useful in methods of preventionand treatment of synucleinopathic and amyloidogenic disease. Optionally,these fragments can be used in combination with an adjuvant.

Alpha synuclein was originally identified in human brains as theprecursor protein of the non-β-amyloid component of (NAC) of AD plaques.(Uéda et al., Proc. Natl. Acad. Sci. U.S.A. 90 (23):11282-11286 (1993).Alpha-SN, also termed the precursor of the non-Aβ component of ADamyloid (NACP), is a peptide of 140 amino acids. Alpha-SN has the aminoacid sequence:

(SEQ ID NO: 1) MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA(Uéda et al., Ibid.; GenBank accession number: P37840).

The non-Aβ component of AD amyloid (NAC) is derived from alpha-SN. NAC,a highly hydrophobic domain within alpha synuclein, is a peptideconsisting of at least 28 amino acids residues (residues 60-87) (SEQ IDNO: 3) and optionally 35 amino acid residues (residues 61-95) (SEQ IDNO: 2). See FIG. 1. NAC displays a tendency to form a beta-sheetstructure (Iwai, et al., Biochemistry, 34:10139-10145). Jensen et al.have reported NAC has the amino acid sequence:

(SEQ ID NO: 2) EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV(Jensen et al., Biochem. J. 310 (Pt 1): 91-94 (1995); GenBank accessionnumber S56746).

Uéda et al. have reported NAC has the acid sequence:

(SEQ ID NO: 3) KEQVTNVGGAVVTGVTAVAQKTVEGAGS

(Uéda et al., PNAS USA 90:11282-11286 (1993).

Disaggregated alpha-SN or fragments thereof, including NAC, meansmonomeric peptide units. Disaggregated alpha-SN or fragments thereof aregenerally soluble, and are capable of self-aggregating to form solubleoligomers. Oligomers of alpha-SN and fragments thereof are usuallysoluble and exist predominantly as alpha-helices. Monomeric alpha-SN maybe prepared in vitro by dissolving lyophilized peptide in neat DMSO withsonication. The resulting solution is centrifuged to remove anyinsoluble particulates. Aggregated alpha-SN or fragments thereof,including NAC, means oligomers of alpha-SN or fragments thereof whichhave associate into insoluble beta-sheet assemblies. Aggregated alpha-SNor fragments thereof, including NAC, means also means fibrillarpolymers. Fibrils are usually insoluble. Some antibodies bind eithersoluble alpha-SN or fragments thereof or aggregated alpha-SN orfragments thereof. Some antibodies bind to oligomers of alpha-synucleinmore strongly than to monomeric forms or fibrillar forms. Someantibodies bind both soluble and aggregated alpha-SN or fragmentsthereof, and optionally oligomeric forms as well.

Alpha-SN, the principal component of the Lewy bodies characteristic ofPD, and epitopic fragments thereof, such as, for example, NAC, orfragments other than NAC, such as fragments at or near the N-terminus orat or near the C-terminus can be used to induce an immunogenic response.Preferably such fragments comprise four or more amino acids of alpha-SNor analog thereof.

Other components of Lewy bodies, for example, synphilin-1, Parkin,ubiquitin, neurofilament, beta-crystallin, and epitopic fragmentsthereof can also be used to induce an immunogenic response.

As noted, certain preferred fragments of alpha-synuclein are from theC-terminal region of the molecule. Such fragments lack residues 1-69 ofhuman alpha-synuclein. Immunization with such fragments generates animmunogenic response comprising antibodies to one or more epitopeswithin residues 70-140 of human alpha-synuclein. Some active fragmentsinclude an epitope at or near the C-terminus of alpha-SN (e.g., withinamino acids 70-140, 100-140, 120-140, 130-140, or 135-140). Some activefragments include an epitope at or near the region recognized bymonoclonal antibody 8A5. Some active fragments include an epitope at ornear the region recognized by monoclonal antibody 9E4 (e.g., withinamino acids 90-140, 98-140, 108-136, 110-130 or 118-126). Some activefragments include an epitope at or near the region recognized bymonoclonal antibody 1H7 (e.g., within amino acids 70-119, 80-109,88-101, or 91-99). In some active fragments, the C terminal residue ofthe epitope is the C-terminal residue of alpha-SN.

Some fragments of alpha synuclein generate antibodies specificallybinding to an epitope within one or more of: SN83-101, SN107-125,SN110-128, SN 124-140, SN 110-130, SN 85-105, SN 118-126 and SN 91-99 ofhuman alpha synuclein. Some fragments generate antibodies exclusivelyspecifically binding within one of the above fragments. For example, thefragment SN83-101 begins at residue 83 and ends at residues 101 ofalpha-synuclein and generates only antibodies specifically binding toSN83-101. Immunogenic C-terminal region fragments include SN 85-99, SN109-123, SN 112-126 and SN126-138 (as shown in FIG. 8), SN 110-130, SN85-105 and other fragments differing from one of these by up to twoadditional or fewer amino acids at either end. Another preferredfragment SN83-140, which includes all of these epitopes.

Some fragments have no more than 40, or no more than 30, contiguousresidues in total from alpha synuclein. Some such fragments comprise SN125-140, SN130-140, SN 87-97, SN111-121, SN114-124 or SN128-136. Somefragments have a total of 5-20, 5-25 or 5-30 contiguous amino acids fromthe C-terminal half of alpha synuclein (i.e., residues 70-140). Somefragments have 5-20 contiguous amino acids from between positions120-140 of alpha synuclein. Particularly preferred fragments includeSN124-140, SN125-140, SN126-140, SN127-140, SN128-140, SN 129-140,SN130-140, SN131-140, SN132-140, SN133-140, SN134-140, SN135-140,SN136-140, SN137-140, SN124-139, SN125-139, SN126-139, SN127-139,SN128-139, SN124-139, SN125-139, SN126-139, SN127-139, SN128-139, SN129-139, SN130-139, SN131-139, SN132-139, SN133-139, SN134-139,SN135-139, SN136-139, SN137-139, SN124-138, SN124-138, SN125-138,SN126-138, SN127-138, SN128-138, SN 129-138, SN130-138, SN131-138,SN132-138, SN133-138, SN134-138, SN135-138, SN136-138, S SN124-137,SN125-137, SN126-137, SN127-137, SN128-137, SN 129-137, SN130-137,SN131-137, SN132-137, SN133-137, SN134-137, SN135-137, SN124-136,SN125-136, SN126-136, SN127-136, SN128-136, SN 129-136, SN130-136,SN131-136, SN132-136, SN133-136, and SN134-136.

Some fragments have 5-20 contiguous amino acids from between positions118-126, 108-136, and 98-140 of alpha synuclein. Some fragments have5-20 contiguous amino acids from between positions 70-99, 91-131, 136,or 91-99 of alpha synuclein.

As shown in Examples IX and X, administration of 6H7, an antibody thatrecognizes the amino terminus of alpha-synuclein, or of 8A5, an antibodythat recognizes the carboxy-terminus of alpha-synuclein, or 9E4, anantibody that recognizes an epitope in the C-terminal region ofsynuclein, reduced alpha-synuclein aggregation in the brain oftransgenic mice over-expressing human alpha-synuclein. It is expectedthat immunization with alpha-synuclein fragments comprising sequences ator near the alpha-synuclein terminal regions will similarly result insuch clearing of aggregates and/or prevent the formation of aggregates.

Other fragments of alpha-synuclein useful for effecting prophylaxis ortreating a disease characterized by Lewy bodies or alpha-synucleinaggregation in the brain (e.g., Parkinson's Disease) are from theN-terminal region of the molecule. Immunization with the fragmentsgenerates an immunogenic response comprising antibodies to one or moreepitopes within residues 1-20 or, in some cases, one or more epitopeswithin residues 1-10. As shown in Example IX, administration of 6H7, anantibody that recognizes the amino terminus of alpha-synuclein, reducedalpha-synuclein aggregation in the brain of transgenic miceover-expressing human alpha-synuclein. It is expected that immunizationwith alpha-synuclein fragments comprising the alpha-synuclein aminoterminal region will similarly result in such clearing of aggregatesand/or prevent the formation of aggregates.

Thus, in an aspect the invention provides a method of effectingprophylaxis or treating a disease characterized by Lewy bodies oralpha-synuclein aggregation in the brain by administering to a patienthaving or at risk of the disease a polypeptide comprising an immunogenicfragment of alpha-synuclein effective to induce an immunogenic responsecomprising antibodies that specifically bind to an epitope withinresidues 1-40, residues 1-20, or residues 1-10 of human alpha-synuclein,residues being numbered according to SEQ ID NO:1. In one embodiment theimmunogenic fragment of alpha-synuclein is free of residues 70-140 ofalpha synuclein. In one embodiment the immunogenic fragment is free ofresidues 41-140 of alpha-synuclein. In one embodiment the immunogenicfragment is free of residues 25-140 of alpha-synuclein.

Suitable immunogens for effecting prophylaxis or treating a diseasecharacterized by Lewy bodies or alpha-synuclein aggregation in the braininclude, but are not limited to, fragments comprising from 5 to 20contiguous amino acid residues from the amino terminus of alphasynuclein. In a preferred embodiment the fragment comprises the first(amino-terminal) residue of alpha synuclein. Thus, exemplary fragmentsinclude the sequence of residues 1 to N_(a) of SEQ ID NO.: 1, whereN_(a) is 5 to 20 (e.g., MDVFMKGLSKAKE GVVAAAE; MDVFMKG LSKAKEGVVAAA;MDVFMKGLSKAKEGVVAA; MDVFMKGLSKAKE GVVA; MDVFMKGLSKAKEGVV;MDVFMKGLSKAKEGV; MDVFMKGLSKAKEG; MDVFMKGLSKAK; MDVFMKGLSKA; MDVFMKGLSK;MDVFMKGLS; MDVFMKGL; MDVFMKG; MDVFMK; and MDVFM. In other preferredembodiments, the fragment does not comprises the amino-terminal residueof alpha synuclein but comprises at the second and/or third residue ofalpha synuclein. Thus, exemplary fragments have the sequence of residues2 to N_(b), and 3 to N_(c) of SEQ ID NO.: 1, where N_(b) is 6 to 21 andN_(c) is 7 to 22 (e.g., DVFMKGLSKAKEGVVAAAEK; DVFM KGLSKAKEGVVAAAE;DVFMKGLSKAKEGVVAAA; DVFMKGLSKAKEGVVAA; DVFMKGLSKAKEGVVA;DVFMKGLSKAKEGVV; DVFMKGLSKAKEGV; DVFMKGLSKAKEG; DVFMKGLSKAK; DVFMKGLSKA;DVFMKGLSK; DVFMKGLS; DVFMKGL; DVFMKG; DVFMK, VFMKGLSKAKEGVVAAAEKT;VFMKGLSKAKEGVVAAAEK; VFMKGLSKAKEGVVAAAE; VFMKGL SKAKEGVVAAA;VFMKGLSKAKEGVVAA; VFMKGLSKAKEGVVA; VFMKGLSKAKEGVV; VFMKGLSKAKEGV;VFMKGLSKAKEG; VFMKGLSKAK; VFMKGLSKA; VFMKGLSK; VFMKGLS; VFMKGL; andVFMKG). As discussed below, the aforementioned fragments may be linkedto a carrier molecule (e.g., a conjugate or fusion protein, see Sec.II(3)). Alternatively, as discussed below, an aforementioned fragmentmay be administered by vaccinating the subject with a nucleic acidencoding the fragment (see Sec. II(4)).

Other fragments of alpha-synuclein useful for effecting prophylaxis ortreating a disease characterized by Lewy bodies or alpha-synucleinaggregation in the brain (e.g., Parkinson's Disease) are from the regionnear serine 129 of alpha-SN. Immunization with fragments including thisresidue, in its phosphorylated form (e.g., SN 124-134 with Ser129phosphorylated generates an immunogenic response comprising antibodiesto one or more epitopes including phosphoSer129. Some active fragmentsinduce antibodies that recognize an epitope at or near the regionrecognized by monoclonal antibody 11A5.

For ease of reference, alpha-SN fragments and epitopes can be referredto based on their position in the molecule, for example and withoutlimitation, fragments containing the alpha-SN N-terminal amino acid,fragments containing the C-terminal amino acid, NAC fragments asdescribed above, fragments containing neither the N-terminal amino acidor the C-terminal amino acid, fragments from the C-terminal half ofalpha-SN, fragments from the N-terminal half of alpha-SN, fragmentswithin the N-terminal 40 residues of alpha-SN. In certain embodimentsfragments can contain from 5 to 100 contiguous residues of alpha-SN, forexample in the range bounded by an lower limit of 5, 6, 7, 8, 9, 10, 11,12, 15, 20, 25, 30 or 40 contiguous residues and an upper limit of 10,12, 15, 20, 25, 30, 35, 40, 50, or 100 contiguous residues, where theupper limit is higher than the lower limit. Preferably the fragmentcontains at least 5, at least 8, at least 10, or at least 15 or at least20 contiguous residues of alpha-SN.

Reference to alpha-SN includes the natural human amino acid sequencesindicated above as well as analogs including allelic, species andinduced variants, full-length forms and immunogenic fragments thereof.Human alpha synuclein, meaning a protein having the same sequence ofamino acids as SEQ ID NO:1 or allelic variants thereof, is preferred inall embodiments. Analogs typically differ from naturally occurringpeptides at one, two or a few positions, often by virtue of conservativesubstitutions. Analogs typically exhibit at least 80 or 90% sequenceidentity with natural peptides. Positions of amino acids in analogs ofnatural alpha synuclein are assigned the numbers of corresponding aminoacids in natural alpha synuclein when the analog and natural alphasynuclein are maximally aligned. Some analogs also include unnaturalamino acids or modifications of N or C terminal amino acids at one, twoor a few positions. For example, the natural glutamic acid residue atposition 139 of alpha-SN can be replaced with iso-aspartic acid.Examples of unnatural amino acids are D, alpha, alpha-disubstitutedamino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,gamma-carboxyglutamate, epsilon-N,N,N-trimethyllysine,epsilon-N-acetyllysine, 0-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,omega-N-methylarginine, beta-alanine, ornithine, norleucine, norvaline,hydroxproline, thyroxine, gamma-amino butyric acid, homoserine,citrulline, and isoaspartic acid. Therapeutic agents also includeanalogs of alpha-SN fragments. Some therapeutic agents of the inventionare all-D peptides, e.g., all-D alpha-SN or all-D NAC, and of all-Dpeptide analogs. Analogs specifically bind to a polyclonal population ofantibodies to natural human alpha synuclein. Fragments and analogs canbe screened for prophylactic or therapeutic efficacy in transgenicanimal models in comparison with untreated or placebo controls asdescribed below.

Alpha-SN, its fragments, and analogs can be synthesized by solid phasepeptide synthesis or recombinant expression, or can be obtained fromnatural sources. Automatic peptide synthesizers are commerciallyavailable from numerous suppliers, such as Applied Biosystems, FosterCity, Calif. Recombinant expression can be in bacteria, such as E. coli,yeast, insect cells or mammalian cells. Procedures for recombinantexpression are described by Sambrook et al., Molecular Cloning: ALaboratory Manual (C.S.H.P. Press, NY 2d ed., 1989). Some forms ofalpha-SN peptide are also available commercially, for example, at BACHEMand American Peptide Company, Inc.

Therapeutic agents also include longer polypeptides that include, forexample, an active fragment of alpha-SN peptide, together with otheramino acids. For example, preferred agents include fusion proteinscomprising a segment of alpha-SN fused to a heterologous amino acidsequence that induces a helper T-cell response against the heterologousamino acid sequence and thereby a B-cell response against the alpha-SNsegment. Such polypeptides can be screened for prophylactic ortherapeutic efficacy in animal models in comparison with untreated orplacebo controls as described below. The alpha-SN peptide, analog,active fragment or other polypeptide can be administered in associatedor multimeric form or in dissociated form therapeutic agents alsoinclude multimers of monomeric immunogenic agents. The therapeuticagents of the invention may include polylysine sequences.

In a further variation, an immunogenic peptide, such as a fragment ofalpha-SN, can be presented by a virus or bacteria as part of animmunogenic composition. A nucleic acid encoding the immunogenic peptideis incorporated into a genome or episome of the virus or bacteria.Optionally, the nucleic acid is incorporated in such a manner that theimmunogenic peptide is expressed as a secreted protein or as a fusionprotein with an outer surface protein of a virus or a transmembraneprotein of bacteria so that the peptide is displayed. Viruses orbacteria used in such methods should be nonpathogenic or attenuated.Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitisvirus and other alpha viruses, vesicular stomatitis virus, and otherrhabdo viruses, vaccinia and fowl pox. Suitable bacteria includeSalmonella and Shigella. Fusion of an immunogenic peptide to HBsAg ofHBV is particularly suitable.

Therapeutic agents also include peptides and other compounds that do notnecessarily have a significant amino acid sequence similarity withalpha-SN but nevertheless serve as mimetics of alpha-SN and induce asimilar immune response. For example, any peptides and proteins formingbeta-pleated sheets can be screened for suitability. Anti-idiotypicantibodies against monoclonal antibodies to alpha-SN or other Lewy bodycomponents can also be used. Such anti-Id antibodies mimic the antigenand generate an immune response to it (see Essential Immunology, Roited., Blackwell Scientific Publications, Palo Alto, Calif. 6th ed., p.181). Agents other than alpha-SN should induce an immunogenic responseagainst one or more of the preferred segments of alpha-SN listed above(e.g., NAC). Preferably, such agents induce an immunogenic response thatis specifically directed to one of these segments without being directedto other segments of alpha-SN.

Random libraries of peptides or other compounds can also be screened forsuitability. Combinatorial libraries can be produced for many types ofcompounds that can be synthesized in a step-by-step fashion. Suchcompounds include polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines and oligocarbamates. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in Affymax, WO 95/12608, Affymax, WO 93/06121, ColumbiaUniversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated herein by reference for allpurposes). Peptide libraries can also be generated by phage displaymethods. See, e.g., Devlin, WO 91/18980.

Combinatorial libraries and other compounds are initially screened forsuitability by determining their capacity to bind to antibodies orlymphocytes (B or T) known to be specific for alpha-SN or other Lewybody components. For example, initial screens can be performed with anypolyclonal sera or monoclonal antibody to alpha-SN or a fragmentthereof. The libraries are preferably screened for capacity to bind toantibodies that specifically bind to an epitope within residues 1-20 or70-140 of human alpha synuclein. Compounds can then be screened forspecific binding to a specific epitope within alpha-SN (e.g., SN 1-20,SN 83-101, SN107-125, SN110-128, SN124-140, SN 118-126 and SN 91-99 ofalpha synuclein). Compounds can be tested by the same proceduresdescribed for mapping antibody epitope specificities. Compoundsidentified by such screens are then further analyzed for capacity toinduce antibodies or reactive lymphocytes to alpha-SN or fragmentsthereof. For example, multiple dilutions of sera can be tested onmicrotiter plates that have been precoated with alpha-SN or a fragmentthereof and a standard ELISA can be performed to test for reactiveantibodies to alpha-SN or the fragment. Compounds can then be tested forprophylactic and therapeutic efficacy in transgenic animals predisposedto a disease associated with the presence of Lewy body, as described inthe Examples. Such animals include, for example, transgenic mice overexpressing alpha-SN or mutant thereof (e.g., alanine to threoninesubstitution at position 53) as described, e.g., in WO 98/59050,Masliah, et al., Science 287: 1265-1269 (2000), and van der Putter, etal., J. Neuroscience 20: 6025-6029 (2000), or such transgenic mice thatalso over express APP or a mutant thereof. Particularly preferred aresuch synuclein transgenic mice bearing a 717 mutation of APP describedby Games et al., Nature 373, 523 (1995) and mice bearing a 670/671Swedish mutation of APP such as described by McConlogue et al., U.S.Pat. No. 5,612,486 and Hsiao et al., Science 274, 99 (1996); Staufenbielet al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997);Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292(1997); Borchelt et al., Neuron 19, 939-945 (1997)). Examples of suchsynuclein/APP transgenic animals are provided in WO 01/60794. Additionalanimal models of PD include 6-hydroxydopamine, rotenone, and1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal models (M.Flint Beal, Nature Reviews Neuroscience 2:325-334 (2001)). The samescreening approach can be used on other potential analogs of alpha-SNand longer peptides including fragments of alpha-SN, described above andother Lewy body components and analog or fragments thereof.

As described herein, administration of antibodies recognizing epitopesin the amino terminal and carboxy terminal regions of alpha-synuclein(i.e., 8A5 and 6H7) reduced alpha-synuclein aggregates in the brains oftransgenic mice over-expressing human alpha-synuclein (see, e.g.,Example IX). Based in part on this discovery, it is contemplated thatinducing an immune response against epitopes at both termini ofalpha-synuclein will have advantages in prophylaxis and therapy. Thus,in one aspect, the invention provides a method for prophylaxis ortreatment of a disease characterized by Lewy bodies or alpha-synucleinaggregation in the brain by inducing an immunogenic response comprisingantibodies that specifically bind to an epitope within residues 1-20, oralternatively residues 1-10, of human alpha-synuclein and an epitopewithin residues 70-140 of human alpha-synuclein. In a preferredembodiment the immunogenic response comprises antibodies thatspecifically bind to an epitope within residues 1-20 of humanalpha-synuclein and residues 120-140 of human alpha-synuclein. An immuneresponse that comprises antibodies against epitopes at both terminalregions of the protein can be referred to as a “dual” immune response. Adual immune response can be induced in a number of ways and the presentinvention is not limited to any particular method of initiating such aresponse.

A dual immune response can be induced by vaccination with a singlepolypeptide that is effective to induce an immunogenic responseincluding antibodies that specifically bind to an epitope withinresidues 1-20 of human alpha-synuclein and antibodies that specificallybind to an epitope within residues 70-140 of human alpha-synuclein. Inpreferred embodiments the antibodies specifically bind to an epitopewithin residues 1-10 and/or within residues 120-140 of humanalpha-synuclein. A polypeptide lacking at least residues 25-69 of humanalpha-synuclein, at least residues 30-110 of human alpha-synuclein, orat least residues 21-119 of human alpha-synuclein can be used. When suchpolypeptides are used the immunogenic response does not includeantibodies that specifically bind to an epitope within residues 25-69 ofhuman alpha-synuclein.

A dual immune response can also be induced by vaccination in combinationof two (or more) polypeptides where one polypeptide induces animmunogenic response including antibodies that specifically bind to anepitope within residues 1-20 of human alpha-synuclein and antibodiesthat specifically bind to an epitope within residues 70-140 of humanalpha-synuclein. In preferred embodiments the antibodies specificallybind to an epitope within residues 1-10 and/or within residues 120-140of human alpha-synuclein. Thus, treatment or prophylaxis can be effectedby administering a first immunogenic fragment of alpha-synuclein thatinduces an immunogenic response comprising antibodies that specificallybind to an epitope within residues 1-20 of human alpha-synuclein and asecond immunogenic fragment that induces an immunogenic responsecomprising antibodies that specifically bind to an epitope withinresidues 70-140 (or 120-140) of human alpha-synuclein. Fragments ofhuman alpha-synuclein can be administered in combination, as discussedabove (e.g., administering as a fusion protein or conjugate, in aco-formulation, or in the same course of therapy).

The invention provides compositions useful for initiating an immuneresponse against epitopes at either or both termini of alpha-synuclein.Compositions include dosage forms and formulations containing two ormore polypeptides, such as described above, where one polypeptideinduces an immunogenic response including antibodies that specificallybind to an epitope within residues 1-20 of human alpha-synuclein andantibodies that specifically bind to an epitope within residues 70-140of human alpha-synuclein. In preferred embodiments the polypeptidesinduce antibodies that specifically bind to an epitope within residues1-10 and/or within residues 120-140 of human alpha-synuclein. Exemplaryformulations (suitable for co-formulating polypeptides) are known in theart and include those described below in Section VII (“TreatmentRegimes”).

The invention also provides kits for initiating an immune responseagainst epitopes at both termini of alpha-synuclein. The kits includetwo or more agents that induce an immunogenic response includingantibodies that specifically bind to an epitope within residues 1-20 ofhuman alpha-synuclein and antibodies that specifically bind to anepitope within residues 70-140 of human alpha-synuclein. The agents canbe combined in a single preparation for simultaneous use. The agents canoccupy separate containers (e.g., vials, syringes, tubes, or the like)each containing a different polypeptide for simultaneous, sequential orseparate use. These agents of the invention can optionally beadministered in combination with other agents that are at least partlyeffective in treatment of Lewy Body Disease. Kits can also includeagents that increase passage of the agents of the invention across theblood-brain barrier, other adjuvants and materials for administration tothe patient.

2. Agents for Passive Immune Response

Therapeutic agents of the invention also include antibodies thatspecifically bind to alpha-SN or other components of Lewy bodies. Thisinvention also provides antibodies that specifically bind to asynuclein-NAC component of an amyloid plaque. Antibodies immunoreactivefor alpha-SN are known (see, for example, Arima, et al., Brian Res. 808:93-100 (1998); Crowther et al., Neuroscience Lett. 292: 128-130 (2000);Spillantini, et al. Nature 388: 839-840 (1997). Such antibodies can bemonoclonal or polyclonal. Some such antibodies bind specifically toinsoluble aggregates of alpha-SN without specifically binding to thesoluble monomeric form. Some specifically bind specifically to thesoluble monomeric form without binding to the insoluble aggregated form.Some specifically bind to both aggregated and soluble monomeric forms.Some such antibodies specifically bind to a naturally occurring shortform of alpha-SN (e.g., NAC) without binding to a naturally occurringfull length alpha-SN. Some antibodies specifically bind to a long formwithout binding to a short form. Some antibodies specifically bind toalpha-SN without binding to other components of LBs. Some antibodiesspecifically bind to alpha-SN without specifically binding to othercomponents of amyloid plaques. See U.S. patent publicationUS20060259986A1 and PCT patent publication WO 05/013889, which areincorporated herein by reference for all purposes, provides end-specificantibodies that specifically bind to fragments of alpha-synucleinwithout specifically binding to intact alpha-synuclein per se. Theseantibodies are useful in methods of prevention and treatment ofsynucleinopathic and amyloidogenic disease.

In experiments carried out in support of the invention, a predictive exvivo assay (Example VII) was used to test clearing of an antibody thatspecifically binds to a synuclein-NAC. An antibody to NAC was contactedwith a brain tissue sample containing amyloid plaques and microglialcells. Rabbit serum was used as a control. Subsequent monitoring showeda marked reduction in the number and size of plaques indicative ofclearing activity of the antibody.

From these data, it is apparent that amyloid plaque load associated withAlzheimer's disease and other amyloid diseases can be greatly diminishedby administration of immune reagents directed against epitopes of NAC,which are effective to reduce amyloid plaque load. It is furtherunderstood that a wide variety of antibodies can be used in suchcompositions. As discussed above, U.S. patent publicationUS20060259986A1 and PCT patent publication WO 05/013889, providesend-specific antibodies that specifically bind to fragments ofalpha-synuclein without specifically binding to intact alpha-synucleinper se.

Antibodies used in therapeutic methods usually have an intact constantregion or at least sufficient of the constant region to interact with anFc receptor. Human isotype IgG1 is preferred because of it havinghighest affinity of human isotypes for the FcRI receptor on phagocyticcells. Bispecific Fab fragments can also be used, in which one arm ofthe antibody has specificity for alpha-SN, and the other for an Fcreceptor. Some antibodies bind to alpha-SN, optionally in a denaturedform, such as when treated with SDS, with a binding affinity greaterthan or equal to about 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Some antibodiesof the invention specifically bind to human alpha synuclein in synapsesor neuronal cell bodies as determined by immunocytochemistry.

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of alpha-SN. However,polyclonal sera can be specific to a particular segment of alpha-SN,such as NAC. Polyclonal sera that is specific for a particular segmentcontains antibodies that specifically bind to that segment and lacksantibodies that specifically bind to other segments of alpha-SN.Monoclonal antibodies bind to a specific epitope within alpha-SN thatcan be a conformational or nonconformational epitope. Nonconformationalepitopes remain present when alpha-SN is denatured with SDS.Prophylactic and therapeutic efficacy of antibodies can be tested usingthe transgenic animal model procedures described in the Examples. somemonoclonal antibodies bind to an epitope within NAC. In some methods,multiple monoclonal antibodies having binding specificities to differentepitopes are used. Such antibodies can be administered sequentially orsimultaneously. Antibodies to Lewy body components other than alpha-SNcan also be used. For example, antibodies can be directed toneurofilament, ubiquitin, or synphilin. Therapeutic agents also includeantibodies raised against analogs of alpha-SN and fragments thereof.Some therapeutic agents of the invention are all-D peptides, e.g., all-Dalpha-SN or all-D NAC.

When an antibody is said to bind to an epitope within specifiedresidues, such as alpha-SN 1-5, for example, what is meant is that theantibody specifically binds to a polypeptide containing the specifiedresidues (i.e., alpha-SN 1-5 in this an example). Such an antibody doesnot necessarily contact every residue within alpha-SN 1-5. Nor doesevery single amino acid substitution or deletion with in alpha-SN1-5necessarily significantly affect binding affinity. Epitope specificityof an antibody can be determined, for example, by forming a phagedisplay library in which different members display differentsubsequences of alpha-SN. The phage display library is then selected formembers specifically binding to an antibody under test. A family ofsequences is isolated. Typically, such a family contains a common coresequence, and varying lengths of flanking sequences in differentmembers. The shortest core sequence showing specific binding to theantibody defines the epitope bound by the antibody. Antibodies can alsobe tested for epitope specificity in a competition assay with anantibody whose epitope specificity has already been determined.

Some antibodies of the invention specifically binds to an epitope withinNAC. Some antibodies specifically bind to an epitope within a22-kilodalton glycosylated form of synuclein, e.g., P22-synuclein (H.Shimura et al., Science 2001 Jul. 13:293(5528):224-5).

Some antibodies of the invention bind to an epitope at the N-terminus ofalpha-SN (for exampe, an epitope within amino acids 1-20 or amino acids1-10 of alpha-synuclein as numbered according to SEQ ID NO:1). Someantibodies bind to an epitope in which the N-terminal residue of theepitope is the N-terminal residue of full-length alpha-SN. Suchantibodies do not bind to deletion mutants of alpha synuclein in whichresidue 1 is missing. Some such antibodies do not bind to full-lengthalpha synuclein in which the N-terminal amino acid is joined to aheterologous polypeptide. Some antibodies of the invention specificallybind to an epitope within residues 1-69 or residues 1-20 of humanalpha-synuclein. Some antibodies specifically bind to an epitope withinresidues 1-20 of human alpha-synuclein. Some antibodies specificallybind to an epitope with a segment of human alpha-synuclein selected fromresidues 1 to N_(a) of SEQ ID NO.: 1, where N_(a) is 5 to 20; residues 2to N_(b) of SEQ ID NO.: 1, where N_(b) is 6 to 21; or residues 3-N_(c)of SEQ ID NO.: 1 where N_(c) is 7 to 22. Some antibodies bind to anepitope within a segment of human alpha synuclein selected from thegroup consisting of consisting of SN1-5, SN1-6, SN1-7, SN1-8, SN1-9,SN1-10, SN1-11, SN1-12, SN1-13, SN1-14 SN1-15, SN1-16, SN1-17, SN1-18,SN1-19, and SN1-20.

Some antibodies binds to an epitope at or near the C-terminus ofalpha-SN (e.g., within amino acids 70-140, 100-140, 120-140, 130-140 or135-140). Some antibodies bind to an epitope in which the C-terminalresidue of the epitope is the C-terminal residue of (full-length)alpha-SN. Such antibodies do not bind to deletion mutants of alphasynuclein in which residue 140 is missing. Some such antibodies do notbind to full-length alpha synuclein in which the C-terminal amino acidis joined to a heterologous polypeptide. In some methods, the antibodyspecifically binds to NAC without binding to full length alpha-SN.

Some antibodies of the invention specifically bind to an epitope withinresidues 70-140 or 83-140 of human alpha synuclein. Some antibodiesspecifically bind to an epitope within residues 120-140 of humanalpha-synuclein. Some antibodies specifically bind to an epitope with asegment of human alpha-synuclein selected from 83-101, 107-125, 110-128and 124-140. Some antibodies bind to an epitope within a segment ofhuman alpha synuclein selected from the group consisting of SN124-140,SN125-140, SN126-140, SN127-140, SN128-140, SN 129-140, SN130-140,SN131-140, SN132-140, SN133-140, SN134-140, SN135-140, SN136-140,SN137-140, SN124-139, SN125-139, SN126-139, SN127-139, SN128-139,SN124-139, SN125-139, SN126-139, SN127-139, SN128-139, SN 129-139,SN130-139, SN131-139, SN132-139, SN133-139, SN134-139, SN135-139,SN136-139, SN137-139, SN124-138, SN124-138, SN125-138, SN126-138,SN127-138, SN128-138, SN 129-138, SN130-138, SN131-138, SN132-138,SN133-138, SN134-138, SN135-138, SN136-138, SN124-137, SN125-137,SN126-137, SN127-137, SN128-137, SN 129-137, SN130-137, SN131-137,SN132-137, SN133-137, SN134-137, SN135-137, SN124-136, SN125-136,SN126-136, SN127-136, SN128-136, SN 129-136, SN130-136, SN131-136,SN132-136, SN133-136, and SN134-136.

Some antibodies bind to an epitope within a segment of humanalpha-synuclein selected from amino acids 90-140, 98-140, 108-136,110-130 or 118-126 of alpha synuclein.

Some antibodies bind to an epitope with a segment of humanalpha-synuclein selected from SN 70-119, 80-109, 88-101, or 91-99 or70-99.

Monoclonal antibodies binding to C-terminal region epitopes preferablybind with high affinity e.g., at least 10⁸, 10⁹ or 10¹⁰ M⁻¹ to humanalpha synuclein.

Some antibodies of the invention specifically recognize alpha-SNphosphorylated at position 129 (serine) and do not specifically bindunphosphorylated alpha-SN. Some antibodies bind to an epitope within thesegment SN 120-130 of human alpha-synuclein selected from, such as SN124-134.

Monoclonal or polyclonal antibodies that specifically bind to apreferred segment of alpha-SN without specifically binding to otherregions of alpha-SN have a number of advantages relative to monoclonalantibodies binding to other regions or polyclonal sera to intactalpha-SN. First, for equal mass dosages, dosages of antibodies thatspecifically bind to preferred segments contain a higher molar dosage ofantibodies effective in clearing amyloid plaques. Second, antibodiesspecifically binding to preferred segments can induce a clearingresponse against LBs without inducing a clearing response against intactalpha-SN, thereby reducing the potential for side effects.

Optionally, antibodies can be screened for prophylactic or therapeuticactivity in transgenic animals of LB disease as described above.Optionally, a collection of antibodies is prescreened for relativebinding to denatured human alpha synuclein or a fragment thereof. Therelative binding affinities can be estimated from relative intensitiesof signal in an immunblot. An antibody having relative binding affinityabove the mean, or preferably the antibody having the highest bindingaffinity tested is selected for further screening in transgenic animals.Similar prescreening can be performed to test antibodies for binding toaggregates of alpha-synuclein in tissue sections by immunocytochemistry.Tissue sections can be obtained from the brain of a diseased patient ora transgenic animal model.

In one embodiment the antibody designated 6H7, or an antibody thatcompetes with 6H7 for specific binding to alpha synuclein is used forpassive immunization. In one embodiment the antibody designated 8A5, oran antibody that competes with 8A5 for specific binding to alphasynuclein is used for passive immunization. In one embodiment theantibody designated 9E4, or an antibody that competes with 9E4 forspecific binding to alpha synuclein is used for passive immunization. Inone embodiment the antibody designated 1H7, or an antibody that competeswith 1H7 for specific binding to alpha synuclein is used for passiveimmunization. In one embodiment the antibody designated 11A5, or anantibody that competes with 11A5 for specific binding to alpha synucleinis used for passive immunization. In some embodiments an aforementionedantibody is used in combination with each other or with other anti-alphasynuclein antibodies.

As described herein, administration of antibodies recognizing epitopesin the amino terminal and carboxy terminal regions of alpha-synuclein(i.e., 8A5 and 6H7) reduced alpha-synuclein aggregates in the brains oftransgenic mice over-expressing human alpha-synuclein (see, e.g.,Example IX). Based in part on this discovery, it is contemplated thatadministration in combination of antibodies that recognize a N-terminalepitope (e.g., as described above) and antibodies that recognize aC-terminal epitope (e.g., as described above) will be particularlyeffective in prophylaxis and therapy. Thus, in one aspect, the inventionprovides a method for prophylaxis or treatment of a diseasecharacterized by Lewy bodies or alpha-synuclein aggregation in the brainby administering in combination to a patient having or at risk of thedisease an effective regime of a first antibody that specifically bindsto an epitope within residues 1-20 of human alpha-synuclein, residuesbeing numbered according to SEQ ID NO:1 and administering a secondantibody that that specifically binds to an epitope within residues70-140 of human alpha-synuclein. Preferably the first antibody binds toan epitope of alpha-synuclein within the sequence of residues 1 to N_(a)of SEQ ID NO.: 1, where N_(a) is 5 to 20; within the sequence ofresidues 2 to N_(b) of SEQ ID NO.: 1, where N_(b) is 6 to 21; and/orwithin the sequence of residues 3 to N_(c) of SEQ ID NO.: 1, and N_(c)is 7 to 22. Preferably the second antibody specifically binds to anepitope within residues 120-140 of human alpha-synuclein. The first andsecond antibodies can be administered simultaneously (e.g.,coformulated), the same day, the same month and/or as part of the samecourse of therapy.

The invention provides compositions for prophylaxis or treatment of adisease characterized by Lewy bodies or alpha-synuclein aggregation inthe brain comprising one or more antibodies that binds at a terminalregion of alpha-synuclein, e.g., having a specificity described above.Compositions include dosage forms and formulations containing two ormore antibodies. Exemplary formulations (suitable for co-formulatingantibodies) are known in the art and include those described below inSection VII (“Treatment Regimes”).

The invention also provides kits for prophylaxis or treatment of adisease characterized by Lewy bodies or alpha-synuclein aggregation inthe brain. The kits include two (or more) antibodies where a firstantibody binds an epitope at the N-terminus of human alpha-synuclein andthe second antibody binds an epitope at the C-terminus of humanalpha-synuclein. The antibodies can be combined in a single preparationor kit for simultaneous use. Alternatively, the antibodies can occupyseparate containers (e.g., vials, syringes, tubes, or the like) in a kitfor simultaneous, sequential or separate use. These antibodies canoptionally be administered in combination with other agents that are atleast partly effective in treatment of Lewy Bidy disease. Kits can alsoinclude agents that increase passage of the antibodies of the inventionacross the blood-brain barrier, other adjuvants and materials foradministration to the patient.

i. General Characteristics of Immunoglobulins

The basic antibody structural unit is known to comprise a tetramer ofsubunits. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); orChothia et al., Nature 342:878-883 (1989).

ii. Production of Nonhuman Antibodies

Chimeric and humanized antibodies have the same or similar bindingspecificity and affinity as a mouse or other nonhuman antibody thatprovides the starting material for construction of a chimeric orhumanized antibody. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). See, Gonzales etal., Minimizing the immunogenicity of antibodies for clinicalapplication, Tumour Biol. 26(1):31-43 (2005). Chimeric antibodies areantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin gene segmentsbelonging to different species. For example, the variable (V) segmentsof the genes from a mouse monoclonal antibody may be joined to humanconstant (C) segments, such as IgG1 and IgG4. Human isotype IgG1 ispreferred. In some methods, the isotype of the antibody is human IgG1.IgM antibodies can also be used in some methods. A typical chimericantibody is thus a hybrid protein consisting of the V or antigen-bindingdomain from a mouse antibody and the C or effector domain from a humanantibody.

Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse-antibody,(referred to as the donor immunoglobulin). See, Queen et al., Proc.Natl. Acad. Sci. USA 86:10029-10033 (1989), WO 90/07861, U.S. Pat. Nos.5,693,762, 5,693,761, 5,585,089, 5,530,101, and Winter, U.S. Pat. No.5,225,539 (each of which is incorporated by reference in its entiretyfor all purposes). The constant region(s), if present, are alsosubstantially or entirely from a human immunoglobulin. The humanvariable domains are usually chosen from human antibodies whoseframework sequences exhibit a high degree of sequence identity with themurine variable region domains from which the CDRs were derived. Theheavy and light chain variable region framework residues can be derivedfrom the same or different human antibody sequences. The human antibodysequences can be the sequences of naturally occurring human antibodiesor can be consensus sequences of several human antibodies. See Carter etal., WO 92/22653. Certain amino acids from the human variable regionframework residues are selected for substitution based on their possibleinfluence on CDR conformation and/or binding to antigen. Investigationof such possible influences is by modeling, examination of thecharacteristics of the amino acids at particular locations, or empiricalobservation of the effects of substitution or mutagenesis of particularamino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,(2) is adjacent to a CDR region,(3) otherwise interacts with a CDR region (e.g. is within about 6 A of aCDR region), or(4) participates in the VL-VH interface.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the mouse donor antibody or from the equivalentpositions of more typical human immunoglobulins. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

Some humanized antibodies comprise complementarity determining regions(CDRs) sequences derived from mouse monoclonal antibody mAb 6H7. Thecell line designated JH17.6H7.1.54.28 producing the antibody 6H7 has theATCC accession number PTA-6910 having been deposited under theprovisions of the Budapest Treaty with the American Type CultureCollection (ATCC, P.O. Box 1549, Manassas, Va. 20108) on Aug. 4, 2005.

Some humanized antibodies comprise complementarity determining regions(CDRs) sequences derived from mouse monoclonal antibody mAb 8A5. Thecell line designated JH4.8A5.25.7.36 producing the antibody 8A5 has theATCC accession number PTA-6909 having been deposited on Aug. 4, 2005.

Some humanized antibodies comprise complementarity determining regions(CDRs) sequences derived from mouse monoclonal antibody mAb 9E4. Thecell line designated JH17.9E4.3.37.1.14.2 producing the antibody 9E4 hasthe ATCC accession number PTA-8221 having been deposited under theprovisions of the Budapest Treaty with the American Type CultureCollection (ATCC, P.O. Box 1549, Manassas, Va. 20108) on Feb. 26, 2007.

Some humanized antibodies comprise complementarity determining regions(CDRs) sequences derived from mouse monoclonal antibody mAb 11A5. Thecell line designated JH22.11A5.6.29.70.54.16.14 producing the antibody11A5 has the ATCC accession number PTA-8222 having been deposited underthe provisions of the Budapest Treaty with the American Type CultureCollection (ATCC, P.O. Box 1549, Manassas, Va. 20108) on Feb. 26, 2007.

Some humanized antibodies comprise complementarity determining regions(CDRs) sequences derived from mouse monoclonal antibody mAb 1H7. Thecell line designated JH17.1H7.4.24.34 producing the antibody 1H7 has theATCC accession number PTA-8220 having been deposited under theprovisions of the Budapest Treaty with the American Type CultureCollection (ATCC, P.O. Box 1549, Manassas, Va. 20108) on Feb. 26, 2007.

As noted above, a number of methods are known for producing chimeric andhumanized antibodies using an antibody-expressing cell line (e.g.,hybridoma). For example, the immunoglobulin variable regions of themouse 8A5 and/or 6H7 and/or 9E4 antibodies can be cloned and sequencedusing well known methods. Likewise, the immunoglobulin variable regionsof the mouse 1H7 or 11A5 antibodies can be cloned and sequenced usingwell known methods. In one method, for illustration and not limitation,the heavy chain variable VH region is cloned by RT-PCR using mRNAprepared from hybridoma cells. Consensus primers are employed to VHregion leader peptide encompassing the translation initiation codon asthe 5′ primer and a g2b constant regions specific 3′ primer. Exemplaryprimers are described in U.S. patent publication US 2005/0009150 bySchenk et al. (hereinafter, “Schenk”). The sequences from multiple,independently-derived clones, can be compared to ensure no changes areintroduced during amplification. The sequence of the VH region can alsobe determined or confirmed by sequencing a VH fragment obtained by 5′RACE RT-PCR methodology and the 3′ g2b specific primer.

The light chain variable VL region of 8A5, 6H7, 9E4, 1H7 or 11A5 can becloned in an analogous manner as the VH region. In one approach, aconsensus primer set designed for amplification of murine VL regions isdesigned to hybridize to the VL region encompassing the translationinitiation codon, and a 3′ primer specific for the murine Ck regiondownstream of the V-J joining region. In a second approach, 5′RACERT-PCR methodology is employed to clone a VL encoding cDNA. Exemplaryprimers are described in Schenk. The cloned sequences are then combinedwith sequences encoding human constant regions.

In one approach, the heavy and light chain variable regions arere-engineered to encode splice donor sequences downstream of therespective VDJ or VJ junctions, and cloned into the mammalian expressionvector, such as pCMV-hyl for the heavy chain, and pCMV-hid for the lightchain. These vectors encode human yl and Ck constant regions as exonicfragments downstream of the inserted variable region cassette. Followingsequence verification, the heavy chain and light chain expressionvectors can be co-transfected into COS cells to produce chimericantibodies. Conditioned media is collected 48 hrs post transfection andassayed by western blot analysis for antibody production or ELISA forantigen binding. The chimeric antibodies are humanized as describedabove.

iii. Human Antibodies

Human antibodies against alpha-SN are provided by a variety oftechniques described below. Some human antibodies are selected bycompetitive binding experiments, or otherwise, to have the same epitopespecificity as a particular mouse antibody, such as one of the mousemonoclonal antibodies described in Examples IX and X. Human antibodiescan also be screened for a particular epitope specificity by using onlya fragment of alpha-SN as the immunogen, and/or by screening antibodiesagainst a collection of deletion mutants of alpha-SN. Human antibodiespreferably have isotype specificity human IgG1.

(1) Trioma Methodology

The basic approach and an exemplary cell fusion partner, SPAZ-4, for usein this approach have been described by Oestberg et al., Hybridoma2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman etal., U.S. Pat. No. 4,634,666 (each of which is incorporated by referencein its entirety for all purposes). The antibody-producing cell linesobtained by this method are called triomas, because they are descendedfrom three cells-two human and one mouse. Initially, a mouse myelomaline is fused with a human B-lymphocyte to obtain anon-antibody-producing xenogeneic hybrid cell, such as the SPAZ-4 cellline described by Oestberg, supra. The xenogeneic cell is then fusedwith an immunized human B-lymphocyte to obtain an antibody-producingtrioma cell line. Triomas have been found to produce antibody morestably than ordinary hybridomas made from human cells.

The immunized B-lymphocytes are obtained from the blood, spleen, lymphnodes or bone marrow of a human donor. If antibodies against a specificantigen or epitope are desired, it is preferable to use that antigen orepitope thereof for immunization. Immunization can be either in vivo orin vitro. For in vivo immunization, B cells are typically isolated froma human immunized with alpha-SN, a fragment thereof, larger polypeptidecontaining alpha-SN or fragment, or an anti-idiotypic antibody to anantibody to alpha-SN. In some methods, B cells are isolated from thesame patient who is ultimately to be administered antibody therapy. Forin vitro immunization, B-lymphocytes are typically exposed to antigenfor a period of 7-14 days in a media such as RPMI-1640 (see Engleman,supra) supplemented with 10% human plasma.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ-4 by well known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees C., forabout 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto alpha-SN or a fragment thereof. Triomas producing human antibodieshaving the desired specificity are subcloned by the limiting dilutiontechnique and grown in vitro in culture medium. The trioma cell linesobtained are then tested for the ability to bind alpha-SN or a fragmentthereof.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into standard mammalian, bacterial or yeast celllines.

(2) Transgenic Non-Human Mammals

Human antibodies against alpha-SN can also be produced from non-humantransgenic mammals having transgenes encoding at least a segment of thehuman immunoglobulin locus. Usually, the endogenous immunoglobulin locusof such transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/1222, U.S. Pat.Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016,5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553(1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741(each of which is incorporated by reference in its entirety for allpurposes). Transgenic mice are particularly suitable. Anti-alpha-SNantibodies are obtained by immunizing a transgenic nonhuman mammal, suchas described by Lonberg or Kucherlapati, supra, with alpha-SN or afragment thereof. Monoclonal antibodies are prepared by, e.g., fusingB-cells from such mammals to suitable myeloma cell lines usingconventional Kohler-Milstein technology. Human polyclonal antibodies canalso be provided in the form of serum from humans immunized with animmunogenic agent. Optionally, such polyclonal antibodies can beconcentrated by affinity purification using alpha-SN or other amyloidpeptide as an affinity reagent.

(3) Phage Display Methods

A further approach for obtaining human anti-alpha-SN antibodies is toscreen a DNA library from human B cells according to the generalprotocol outlined by Huse et al., Science 246:1275-1281 (1989). Asdescribed for trioma methodology, such B cells can be obtained from ahuman immunized with alpha-SN, fragments, longer polypeptides containingalpha-SN or fragments or anti-idiotypic antibodies. Optionally, such Bcells are obtained from a patient who is ultimately to receive antibodytreatment. Antibodies binding to alpha-SN or a fragment thereof areselected. Sequences encoding such antibodies (or binding fragments) arethen cloned and amplified. The protocol described by Huse is renderedmore efficient in combination with phage-display technology. See, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat.Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743 and 5,565,332(each of which is incorporated by reference in its entirety for allpurposes). In these methods, libraries of phage are produced in whichmembers display different antibodies on their outer surfaces. Antibodiesare usually displayed as Fv or Fab fragments. Phage displayingantibodies with a desired specificity are selected by affinityenrichment to an alpha-SN peptide or fragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeWinter, WO 92/20791. In this method, either the heavy or light chainvariable region of the selected murine antibody is used as a startingmaterial. If, for example, a light chain variable region is selected asthe starting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding foralpha-SN (e.g., at least 10⁸ and preferably at least 10⁹ M¹) isselected. The human heavy chain variable region from this phage thenserves as a starting material for constructing a further phage library.In this library, each phage displays the same heavy chain variableregion (i.e., the region identified from the first display library) anda different light chain variable region. The light chain variableregions are obtained from a library of rearranged human variable lightchain regions. Again, phage showing strong specific binding for alpha-SNare selected. These phage display the variable regions of completelyhuman anti-alpha-SN antibodies. These antibodies usually have the sameor similar epitope specificity as the murine starting material.

iv. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized, orhuman antibodies can be linked to at least a portion of a human constantregion. The choice of constant region depends, in part, whetherantibody-dependent complement and/or cellular mediated toxicity isdesired. For example, isotopes IgG1 and IgG3 have complement activityand isotypes IgG2 and IgG4 do not. Choice of isotype can also affectpassage of antibody into the brain. Human isotype IgG1 is preferred.Light chain constant regions can be lambda or kappa. Antibodies can beexpressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′ F(ab′)2, and Fv, or assingle chain antibodies in which heavy and light chain variable domainsare linked through a spacer.

v. Expression of Recombinant Antibodies

Chimeric, humanized and human antibodies are typically produced byrecombinant expression. Recombinant polynucleotide constructs typicallyinclude an expression control sequence operably linked to the codingsequences of antibody chains, including naturally associated orheterologous promoter regions. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g.,ampicillin-resistance or hygromycin-resistance, to permit detection ofthose cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host particularly useful for cloning the DNAsequences of the present invention. Microbes, such as yeast are alsouseful for expression. Saccharomyces is a preferred yeast host, withsuitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, NY, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines, various COS celllines, HeLa cells, L cells, human embryonic kidney cell, and myelomacell lines. Preferably, the cells are nonhuman. Expression vectors forthese cells can include expression control sequences, such as an originof replication, a promoter, an enhancer (Queen et al., Immunol. Rev.89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from endogenous genes, cytomegalovirus,SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J.Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. Nos. 5,741,957, 5,304,489, 5,849,992). Suitable transgenesinclude coding sequences for light and/or heavy chains in operablelinkage with a promoter and enhancer from a mammary gland specific gene,such as casein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment,electroporation, lipofection, biolistics or viral-based transfection canbe used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al., supra). For production of transgenic animals, transgenes can bemicroinjected into fertilized oocytes, or can be incorporated into thegenome of embryonic stem cells, and the nuclei of such cells transferredinto enucleated oocytes.

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

3. Conjugates

Some agents for inducing an immune response contain the appropriateepitope for inducing an immune response against LBs but are too small tobe immunogenic. In this situation, a peptide immunogen can be linked toa suitable carrier molecule to form a conjugate which helps elicit animmune response. Suitable carriers include serum albumins, keyholelimpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin,tetanus toxoid, or a toxoid from other pathogenic bacteria, such asdiphtheria, E. coli, cholera, or H. pylori, or an attenuated toxinderivative. T cell epitopes are also suitable carrier molecules. Someconjugates can be formed by linking agents of the invention to animmunostimulatory polymer molecule (e.g., tripalmitoyl-S-glycerinecysteine (Pam3Cys), mannan (a manose polymer), or glucan (a beta 1->2polymer)), cytokines (e.g., IL-1, IL-1 alpha and beta peptides, IL-2,gamma-INF, IL-10, GM-CSF), and chemokines (e.g., MIP1alpha and beta, andRANTES). Immunogenic agents can also be linked to peptides that enhancetransport across tissues, as described in O'Mahony, WO 97/17613 and WO97/17614. Immunogens may be linked to the carries with or with outspacers amino acids (e.g., gly-gly).

Some conjugates can be formed by linking agents of the invention to atleast one T cell epitope. Some T cell epitopes are promiscuous whileother T cell epitopes are universal. Promiscuous T cell epitopes arecapable of enhancing the induction of T cell immunity in a wide varietyof subjects displaying various HLA types. In contrast to promiscuous Tcell epitopes, universal T cell epitopes are capable of enhancing theinduction of T cell immunity in a large percentage, e.g., at least 75%,of subjects displaying various HLA molecules encoded by different HLA-DRalleles.

A large number of naturally occurring T-cell epitopes exist, such as,tetanus toxoid (e.g., the P2 and P30 epitopes), Hepatitis B surfaceantigen, pertussis, toxoid, measles virus F protein, Chlamydiatrachomitis major outer membrane protein, diphtheria toxoid, Plasmodiumfalciparum circumsporozite T, Plasmodium falciparum CS antigen,Schistosoma mansoni triose phosphate isomersae, Escherichia coli TraT,and Influenza virus hemagluttinin (HA). The immunogenic peptides of theinvention can also be conjugated to the T-cell epitopes described inSinigaglia F. et al., Nature, 336:778-780 (1988); Chicz R. M. et al., J.Exp. Med., 178:27-47 (1993); Hammer J. et al., Cell 74:197-203 (1993);Falk K. et al., Immunogenetics, 39:230-242 (1994); WO 98/23635; and,Southwood S. et al. J. Immunology, 160:3363-3373 (1998) (each of whichis incorporated herein by reference for all purposes). Further examplesinclude:

Influenza Hemagluttinin: (SEQ ID NO: 4) HA₃₀₇₋₃₁₉ PKYVKQNTLKLATMalaria CS: (SEQ ID NO: 5) T3 epitope EKKIAKMEKASSVFNVHepatitis B surface antigen: (SEQ ID NO: 6) HBsAg₁₉₋₂₈ FFLLTRILTIHeat Shock Protein 65: (SEQ ID NO: 7) hsp65₁₅₃₋₁₇₁ DQSIGDLIAEAMDKVGNEGbacille Calmette-Guerin (SEQ ID NO: 8) QVHFQPLPPAVVKL Tetanus toxoid:(SEQ ID NO: 9) TT₈₃₀₋₈₄₄ QYIKANSKFIGITEL Tetanus toxoid: (SEQ ID NO: 10)TT₉₄₇₋₉₆₇ FNNFTVSFWLRVPKVSASHLE HIV gp120 T1: (SEQ ID NO: 11)KQIINMWQEVGKAMYA

Alternatively, the conjugates can be formed by linking agents of theinvention to at least one artificial T-cell epitope capable of binding alarge proportion of MHC Class II molecules, such as the pan DR epitope(“PADRE”). PADRE is described in U.S. Pat. No. 5,736,142, WO 95/07707,and Alexander J et al., Immunity, 1:751-761 (1994) (each of which isincorporated herein by reference for all purposes). A preferred PADREpeptide is AKXVAAWTLKAAA (SEQ ID NO: 12), (common residues bolded)wherein X is preferably cyclohexylalanine, tyrosine or phenylalanine,with cyclohexylalanine being most preferred.

Immunogenic agents can be linked to carriers by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the epsilon-amino on a lysine, or otherfree amino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenicity can be improved through the addition of spacer residues(e.g., Gly-Gly) between the T_(h) epitope and the peptide immunogen ofthe invention. In addition to physically separating the T_(h) epitopefrom the B cell epitope (i.e., the peptide immunogen), the glycineresidues can disrupt any artificial secondary structures created by thejoining of the T_(h) epitope with the peptide immunogen, and therebyeliminate interference between the T and/or B cell responses. Theconformational separation between the helper epitope and the antibodyeliciting domain thus permits more efficient interactions between thepresented immunogen and the appropriate T_(h) and B cells.

To enhance the induction of T cell immunity in a large percentage ofsubjects displaying various HLA types to an agent of the presentinvention, a mixture of conjugates with different T_(h) cell epitopescan be prepared. The mixture may contain a mixture of at least twoconjugates with different T_(h) cell epitopes, a mixture of at leastthree conjugates with different T_(h) cell epitopes, or a mixture of atleast four conjugates with different T_(h) cell epitopes. The mixturemay be administered with an adjuvant.

Immunogenic peptides can also be expressed as fusion proteins withcarriers (i.e., heterologous peptides). The immunogenic peptide can belinked at its amino terminus, its carboxyl terminus, or both to acarrier. Optionally, multiple repeats of the immunogenic peptide can bepresent in the fusion protein. Optionally, an immunogenic peptide can belinked to multiple copies of a heterologous peptide, for example, atboth the N and C termini of the peptide. Some carrier peptides serve toinduce a helper T-cell response against the carrier peptide. The inducedhelper T-cells in turn induce a B-cell response against the immunogenicpeptide linked to the carrier peptide.

Some agents of the invention comprise a fusion protein in which anN-terminal fragment of alpha-SN is linked at its C-terminus to a carrierpeptide. In such agents, the N-terminal residue of the fragment ofalpha-SN constitutes the N-terminal residue of the fusion protein.Accordingly, such fusion proteins are effective in inducing antibodiesthat bind to an epitope that requires the N-terminal residue of alpha-SNto be in free form. Some agents of the invention comprise a plurality ofrepeats of NAC linked at the C-terminus to one or more copy of a carrierpeptide. Some fusion proteins comprise different segments of alpha-SN intandem.

Some agents of the invention comprise a fusion protein in which aC-terminal fragment of alpha-SN is linked at its N-terminus to a carrierpeptide. In such agents, the C-terminal residue of the fragment ofalpha-SN constitutes the C-terminal residue of the fusion protein.Accordingly, such fusion proteins are effective in inducing antibodiesthat bind to an epitope that requires the C-terminal residue of alpha-SNto be in free form. Some agents of the invention comprise a plurality ofrepeats of a C-terminal peptide, such as SN125-140 linked at theN-terminus to one or more copy of a carrier peptide. Some fusionproteins comprise different segments of alpha-SN in tandem.

In some fusion proteins, NAC is fused at its N-terminal end to aheterologous carrier peptide. In some fusion proteins, NAC is fused atits C-terminal end to a heterologous carrier peptide. Some fusionproteins comprise a heterologous peptide linked to the N-terminus orC-terminus of NAC, which is in turn linked to one or more additional NACsegments of alpha-SN in tandem. Some fusion proteins comprise multiplecopies of a C-terminal alpha synuclein peptide, as described above, andmultiple copies of a heterologous peptide interlinked to one another.

In some fusion proteins, a fragment of alpha-SN not including either theC-terminus or N-terminus (e.g., SN110-130, SN 85-105) is fused at itsN-terminal end to a heterologous carrier peptide. In some fusionproteins, the fragment is fused at its C-terminal end to a heterologouscarrier peptide. Some fusion proteins comprise a heterologous peptidelinked to the N-terminus or C-terminus of the fragment, which is in turnlinked to one or more additional fragments of alpha-SN in tandem. Somefusion proteins comprise multiple copies of a alpha synuclein peptide,as described above, and multiple copies of a heterologous peptideinterlinked to one another.

Some examples of fusion proteins suitable for use in the invention areshown below. Some of these fusion proteins comprise segments of alpha-SN(including any of the fragments described above) linked to tetanustoxoid epitopes such as described in U.S. Pat. No. 5,196,512, EP 378,881and EP 427,347. Some fusion proteins comprise segments of alpha-SNlinked to at least one PADRE. Some heterologous peptides are promiscuousT-cell epitopes, while other heterologous peptides are universal T-cellepitopes. In some methods, the agent for administration is simply asingle fusion protein with an alpha-SN segment linked to a heterologoussegment in linear configuration. The therapeutic agents of the inventionmay be represented using a formula. For example, in some methods, theagent is multimer of fusion proteins represented by the formula 2^(x),in which x is an integer from 1-5. Preferably x is 1, 2, or 3, with 2being most preferred. When x is two, such a multimer has four fusionproteins linked in a preferred configuration referred to as MAP4 (seeU.S. Pat. No. 5,229,490).

The MAP4 configuration is shown below, where branched structures areproduced by initiating peptide synthesis at both the N terminal and sidechain amines of lysine. Depending upon the number of times lysine isincorporated into the sequence and allowed to branch, the resultingstructure will present multiple N termini. In this example, fouridentical N termini have been produced on the branched lysine-containingcore. Such multiplicity greatly enhances the responsiveness of cognate Bcells.

Z refers to the NAC peptide, a fragment of the NAC peptide, or otheractive fragment of alpha-SN as described in section I. 2 above. Z mayrepresent more than one active fragment, for example:

Z = alpha-SN 60-72 (NAC region) peptide = (SEQ ID NO: 13)NH2-KEQVTNVCGGAVVT-COOH Z = alpha-SN 73-84 (NAC region) peptide =(SEQ ID NO: 14) NH2-GVTAVAQKTVECG-COOH Z = alpha-SN 102-112 peptide = (SEQ ID NO: 15) NH2-C-amino-heptanoic acid-KNEEGAPCQEG-COOHalpha-SN 128-140 peptide

Other examples of fusion proteins include:

Z-Tetanus toxoid 830-844 in a MAP4 configuration:

(SEQ ID NO: 16) Z-QYIKANSKFIGITELZ-Tetanus toxoid 947-967 in a MAP4 configuration:

(SEQ ID NO: 17) Z-FNNFTVSFWLRVPKVSASHLEZ-Tetanus toxoid₈₃₀₋₈₄₄ in a MAP4 configuration:

(SEQ ID NO: 18) Z-QYIKANSKFIGITELZ-Tetanus toxoid₈₃₀₋₈₄₄+Tetanus toxoid₉₄₇₋₉₆₇ in a linear configuration:

(SEQ ID NO: 19) Z-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE

PADRE peptide (all in linear configurations), wherein X is preferablycyclohexylalanine, tyrosine or phenylalanine, with cyclohexylalaninebeing most preferred-Z:

AKXVAAWTLKAAA(SEQ ID NO: 20)-Z 3Z-PADRE peptide: (SEQ ID NO: 21)Z-Z-Z-AKXVAAWTLKAAA

Further examples of fusion proteins include:

(SEQ ID NO: 22) AKXVAAWTLKAAA- Z-Z-Z-Z (SEQ ID NO: 23) Z-AKXVAAWTLKAAA(SEQ ID NO: 24) Z-ISQAVHAAHAEINEAGR (SEQ ID NO: 25) PKYVKQNTLKLAT- Z-Z-Z(SEQ ID NO: 26) Z-PKYVKQNTLKLAT- Z (SEQ ID NO: 27) Z-Z-Z-PKYVKQNTLKLAT(SEQ ID NO: 28) Z-Z-PKYVKQNTLKLAT (SEQ ID NO: 29)Z-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSK FIGITEL-FNNFTVSFWLRVPKVSASHLE-(SEQ ID NO: 58) Z-Z-Z-Z-QYIKANSKFIGITEL- FNNFTVSFWLRVPKVSASHLE(SEQ ID NO: 30) Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE- (SEQ ID NO: 59)Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE- (SEQ ID NO: 31)ZZ-QYIKANSKFIGITEL  on a 2 branched resin:

(SEQ ID NO: 32) EQVTNVGGAISQAVHAAHAEINEAGR(Synuclein fragment fusion protein  in MAP-4 configuration)

The same or similar carrier proteins and methods of linkage can be usedfor generating immunogens to be used in generation of antibodies againstalpha-SN for use in passive immunization. For example, alpha-SN or afragment linked to a carrier can be administered to a laboratory animalin the production of monoclonal antibodies to alpha-SN.

4. Nucleic Acid Encoding Therapeutic Agents

Immune responses against Lewy bodies can also be induced byadministration of nucleic acids encoding segments of alpha-SN peptide,and fragments thereof, other peptide immunogens, or antibodies and theircomponent chains used for passive immunization. Such nucleic acids canbe DNA or RNA. A nucleic acid segment encoding an immunogen is typicallylinked to regulatory elements, such as a promoter and enhancer thatallow expression of the DNA segment in the intended target cells of apatient. For expression in blood cells, as is desirable for induction ofan immune response, promoter and enhancer elements from light or heavychain immunoglobulin genes or the CMV major intermediate early promoterand enhancer are suitable to direct expression. The linked regulatoryelements and coding sequences are often cloned into a vector. Foradministration of double-chain antibodies, the two chains can be clonedin the same or separate vectors. The nucleic acid encoding therapeuticagents of the invention may also encode at least one T cell epitope. Thedisclosures herein which relates to the use of adjuvants and the use ofapply mutatis mutandis to their use with the nucleic acid encodingtherapeutic agents of the present invention.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3,102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou etal., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519(1996)), Venezuelan equine encephalitis virus (see U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (see WO96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy 6,325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, NucleicAcids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833, and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), (see, e.g., McGee et al., J. Micro Encap.1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346). Such vectorscan further include facilitating agents such as bupivacine (see e.g.,U.S. Pat. No. 5,593,970). DNA can also be administered using a gene gun.See Xiao & Brandsma, supra. The DNA encoding an immunogen isprecipitated onto the surface of microscopic metal beads. Themicroprojectiles are accelerated with a shock wave or expanding heliumgas, and penetrate tissues to a depth of several cell layers. Forexample, The Accel™ Gene Delivery Device manufactured by Agacetus, Inc.Middleton, Wis. is suitable. Alternatively, naked DNA can pass throughskin into the blood stream simply by spotting the DNA onto skin withchemical or mechanical irritation (see WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, and tissue biopsy) or universaldonor hematopoietic stem cells, followed by reimplantation of the cellsinto a patient, usually after selection for cells which haveincorporated the vector.

III. Agents for Inducing Immunogenic Response Against Aβ

Aβ, also known as β-amyloid peptide, or A4 peptide (see U.S. Pat. No.4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun. 120, 1131(1984)), is a peptide of 39-43 amino acids, which is the principalcomponent of characteristic plaques of Alzheimer's disease. Aβ isgenerated by processing of a larger protein APP by two enzymes, termed βand γ secretases (see Hardy, TINS 20, 154 (1997)). Known mutations inAPP associated with Alzheimer's disease occur proximate to the site of βor γ secretase, or within Aβ. For example, position 717 is proximate tothe site of γ-secretase cleavage of APP in its processing to Aβ, andpositions 670/671 are proximate to the site of β-secretase cleavage. Itis believed that the mutations cause AD by interacting with the cleavagereactions by which Aβ is formed so as to increase the amount of the42/43 amino acid form of Aβ generated.

Aβ has the unusual property that it can fix and activate both classicaland alternate complement cascades. In particular, it binds to Clq andultimately to C3bi. This association facilitates binding to macrophagesleading to activation of B cells. In addition, C3bi breaks down furtherand then binds to CR2 on B cells in a T cell dependent manner leading toa 10,000 increase in activation of these cells. This mechanism causes Aβto generate an immune response in excess of that of other antigens.

Aβ has several natural occurring forms. The human forms of Aβ arereferred to as Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43. The sequences of thesepeptides and their relationship to the APP precursor are illustrated byFIG. 1 of Hardy et al., TINS 20, 155-158 (1997). For example, Aβ42 hasthe sequence:

(SEQ ID NO: 33) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

Aβ41, Aβ40 and Aβ39 differ from Aβ42 by the omission of Ala, Ala-Ile,and Ala-Ile-Val respectively from the C-terminal end. Aβ43 differs fromAβ42 by the presence of a Thr residue at the C-terminus.

Analogous agents to those described above for alpha-SN have previouslybeen described for Aβ (see WO 98/25386 and WO 00/72880, both of whichare incorporated herein for all purposes). These agents include Aβ andactive fragments thereof, conjugates of Aβ, and conjugates of Aβ activefragments, antibodies to Aβ and active fragments thereof (e.g., mouse,humanized, human, and chimeric antibodies), and nucleic acids encodingantibody chains. Active fragments from the N-terminal half of Aβ arepreferred. Preferred immunogenic fragments include Aβ1-5, 1-6, 1-7,1-10, 3-7, 1-3, and 1-4. The designation Aβ1-5 for example, indicates afragment including residues 1-5 of Aβ and lacking other residues of Aβ.Fragments beginning at residues 1-3 of Aβ and ending at residues 7-11 ofAβ are particularly preferred.

The disclosures herein which relates to agents inducing an active immuneresponse, agents for inducing a passive immune response, conjugates, andnucleic acids encoding therapeutic agents (see Sections II. 1, 2, 3, and4, above) apply mutatis mutandis to the use of Aβ and fragments thereof.The disclosures herein which relate to agents inducing an active immuneresponse, agents for inducing a passive immune response, conjugates, andnucleic acids encoding therapeutic agents (see Sections II. 1, 2, 3, and4, above) apply mutatis mutandis to the use of Aβ and fragments thereof.The disclosures herein which relate to patients amendable to treatment,and treatment regimes (see Sections IV and V, below) apply mutatismutandis to the use of Aβ and fragments thereof.

Disaggregated Aβ or fragments thereof means monomeric peptide units.Disaggregated Aβ or fragments thereof are generally soluble, and arecapable of self-aggregating to form soluble oligomers. Oligomers of Aβand fragments thereof are usually soluble and exist predominantly asalpha-helices or random coils. Aggregated Aβ or fragments thereof, meansoligomers of alpha-SN or fragments thereof that have associate intoinsoluble beta-sheet assemblies. Aggregated Aβ or fragments thereof,means also means fibrillar polymers. Fibrils are usually insoluble. Someantibodies bind either soluble Aβ or fragments thereof or aggregated Aβor fragments thereof. Some antibodies bind both soluble Aβ or fragmentsthereof and aggregated Aβ or fragments thereof.

Some examples of conjugates include:

AN90549 (Aβ1-7-Tetanus toxoid 830-844 in a MAP4 configuration):(SEQ ID NO: 34) DAEFRHD-QYIKANSKFIGITELAN90550 (Aβ 1-7-Tetanus toxoid 947-967 in a MAP4 configuration):(SEQ ID NO: 35) DAEFRHD-FNNFTVSFWLRVPKVSASHLEAN90542 (Aβ 1-7-Tetanus toxoid 830-844 + 947-967in a linear configuration): (SEQ ID NO: 36)DAEFRHD-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLEAN90576: (Aβ3-9)-Tetanus toxoid 830-844 in a MAP4 configuration):(SEQ ID NO: 37) EFRHDSG-QYIKANSKFIGITEL

PADRE peptide (all in linear configurations), wherein X is preferablycyclohexylalanine, tyrosine or phenylalanine, with cyclohexylalaninebeing most preferred:

AN90562 (PADRE-Aβ1-7): (SEQ ID NO: 38) AKXVAAWTLAAA-DAEFRHDAN90543 (3 PADRE-Aβ1-7): (SEQ ID NO: 39)DAEFRHD-DAEFRHD-DAEFRHD-AKXVAAWTLKAAA

Other examples of fusion proteins (immunogenic epitope of Aβ bolded)include:

(SEQ ID NO: 40) AKXVAAWTLKAAA-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 41)DAEFRHD-AKXVAAWTLKAAA (SEQ ID NO: 42) DAEFRHD-ISQAVHAAHAEINEAGR(SEQ ID NO: 43) FRHDSGY-ISQAVHAAHAEINEAGR (SEQ ID NO: 44)EFRHDSG-ISQAVHAAHAEINEAGR (SEQ ID NO: 45)PKYVKQNTLKLAT-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 46)DAEFRHD-PKYVKQNTLKLAT-DAEFRHD (SEQ ID NO: 47)DAEFRHD-DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO: 48)DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO: 49)DAEFRHD-PKYVKQNTLKLAT-EKKIAKMEKASSVFNVQYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE-DAEFRHD (SEQ ID NO: 50)DAEFRHD-DAEFRHD-DAEFRHD-QYIKANSKFIGITELNNFTV SFWLRVPKVSASHLE(SEQ ID NO: 51) DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE(SEQ ID NO: 52) DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKV SASHLE-DAEFRHD(SEQ ID NO: 53) DAEFRHD-QYIKANSKFIGITEL  on a 2 branched resin.

Preferred monoclonal antibodies bind to an epitope within residues 1-10of Aβ (with the first N terminal residue of natural Aβ designated 1).Some preferred monoclonal antibodies bind to an epitope within aminoacids 1-5, and some to an epitope within 5-10. Some preferred antibodiesbind to epitopes within amino acids 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7. Somepreferred antibodies bind to an epitope starting at resides 1-3 andending at residues 7-11 of Aβ. Other antibodies include those binding toepitopes with residues 13-280 (e.g., monoclonal antibody 266). Preferredantibodies have human IgG1 isotype.

IV. Screening Antibodies for Clearing Activity

The invention provides methods of screening an antibody for activity inclearing a Lewy body or any other antigen, or associated biologicalentity, for which clearing activity is desired. To screen for activityagainst a Lewy body, a tissue sample from a brain of a patient with PDor an animal model having characteristic Parkinson's pathology iscontacted with phagocytic cells bearing an Fc receptor, such asmicroglial cells, and the antibody under test in a medium in vitro. Thephagocytic cells can be a primary culture or a cell line, such as BV-2,C8-B4, or THP-1. In some methods, the components are combined on amicroscope slide to facilitate microscopic monitoring. In some methods,multiple reactions are performed in parallel in the wells of amicrotiter dish. In such a format, a separate miniature microscope slidecan be mounted in the separate wells, or a nonmicroscopic detectionformat, such as ELISA detection of alpha-SN can be used. Preferably, aseries of measurements is made of the amount of Lewy body in the invitro reaction mixture, starting from a baseline value before thereaction has proceeded, and one or more test values during the reaction.The antigen can be detected by staining, for example, with afluorescently labeled antibody to alpha-SN or other components of LBs.The antibody used for staining may or may not be the same as theantibody being tested for clearing activity. A reduction relative tobaseline during the reaction of the LBs indicates that the antibodyunder test has clearing activity. Such antibodies are likely to beuseful in preventing or treating PD and other LBD.

Analogous methods can be used to screen antibodies for activity inclearing other types of biological entities. The assay can be used todetect clearing activity against virtually any kind of biologicalentity. Typically, the biological entity has some role in human oranimal disease. The biological entity can be provided as a tissue sampleor in isolated form. If provided as a tissue sample, the tissue sampleis preferably unfixed to allow ready access to components of the tissuesample and to avoid perturbing the conformation of the componentsincidental to fixing. Examples of tissue samples that can be tested inthis assay include cancerous tissue, precancerous tissue, tissuecontaining benign growths such as warts or moles, tissue infected withpathogenic microorganisms, tissue infiltrated with inflammatory cells,tissue bearing pathological matrices between cells (e.g., fibrinouspericarditis), tissue bearing aberrant antigens, and scar tissue.Examples of isolated biological entities that can be used includealpha-SN, viral antigens or viruses, proteoglycans, antigens of otherpathogenic microorganisms, tumor antigens, and adhesion molecules. Suchantigens can be obtained from natural sources, recombinant expression orchemical synthesis, among other means. The tissue sample or isolatedbiological entity is contacted with phagocytic cells bearing Fcreceptors, such as monocytes or microglial cells, and an antibody to betested in a medium. The antibody can be directed to the biologicalentity under test or to an antigen associated with the entity. In thelatter situation, the object is to test whether the biological entity isvicariously phagocytosed with the antigen. Usually, although notnecessarily, the antibody and biological entity (sometimes with anassociated antigen) are contacted with each other before adding thephagocytic cells. The concentration of the biological entity and/or theassociated antigen, if present, remaining in the medium is thenmonitored. A reduction in the amount or concentration of antigen or theassociated biological entity in the medium indicates the antibody has aclearing response against the antigen and/or associated biologicalentity in conjunction with the phagocytic cells.

Antibodies or other agents can also be screened for activity in clearingLewy bodies using the in vitro assay described in Example II. Neuronalcells transfected with an expression vector expressing synuclein formsynuclein inclusions that can be visualized microscopically. Theactivity of an antibody or other agent in clearing such inclusions canbe determined comparing appearance or level of synuclein in transfectedcells treated with agent with appearance or level of synuclein incontrol cells not treated with the agent. A reduction in size orintensity of synuclein inclusions or a reduction in level of synucleinsignals activity in clearing synuclein. The activity can be monitoredeither by visualizing synuclein inclusions microscopically or by runningcell extracts on a gel and visualizing a synuclein band. As noted inExample 1, section 2, the change in level of synuclein is most marked ifthe extracts are fractionated into cytosolic and membrane fractions, andthe membrane fraction is analyzed.

Antibodies or other agents can also be screened for activity in clearingLewy bodies using the in vivo assay described in Example IX. Briefly, atest antibody is injected into the neocortex of transgenic mice thatoverexpress human α-synuclein and have intraneuronal α-synucleinaggregates. In one approach, the animals used are 4 to 8 month-oldheterozygous transgenic mice overexpressing human wildtype α-synucleinin the brain under the transcriptional control of the PDGF promoter (seeMasliah, 2000, Science 287:1265-69). The test antibody and controls(e.g., irrelevant, isotype-matched control antibodies) are dissolved ina suitable solution (e.g., sterile phosphate-buffered-saline solution)for injection into mice. For each mouse, 2 μl of a 2 mg/ml antibodysolution is injected stereotactically under anesthesia into the deeplayers of the parietal neocortex of the right brain hemisphere(ipsilateral side). The left hemispheres (contralateral side) serve asan baseline control for each mouse. Injection sites are sutured and micemonitored until they recovered from anesthesia. Two weeks afterinjection, mice are euthanized, their brains removed and fixed in 4%paraformaldehyde for 48 h, and cut coronally at 40 μm thickness.Sections around the injection site are stained with an α-synucleinantibody (e.g., ELADW-47, recognizing a-synuclein amino acids 115-122).For each section, intraneuronal α-synuclein aggregates are counted in 4microscopic fields (20× objective) around the injection site in theipsilateral hemisphere, and in 4 fields corresponding fields in thecontralateral control hemisphere. For each animal the α-synucleinaggregate counts for two sections are added and the difference betweenthe total α-synuclein aggregate count between the two hemisphere is todetermine the effect of the test antibody on aggregate clearance foreach individual mouse. A reduction in total α-synuclein aggregate countin the treated hemisphere is indicative that the antibodies or otheragent has activity in clearing Lewy bodies. Preferably a reduction of atleast 10% is observed. More preferably a reduction of at least 20%, atleast 40%, at least 60% or at least 80% is observed.

V. Patients Amenable to Anti-Lewy Body Component Treatment Regimes

Patients amenable to treatment include individuals at risk of asynucleinopathic disease but not showing symptoms, as well as patientspresently showing symptoms. Patients amenable to treatment also includeindividuals at risk of disease of a LBD but not showing symptoms, aswell as patients presently showing symptoms. Such diseases includeParkinson's disease (including idiopathic Parkinson's disease), DLB,DLBD, LBVAD, pure autonomic failure, Lewy body dysphagia, incidentalLBD, inherited LBD (e.g., mutations of the alpha-SN gene, PARK3 andPARK4) and multiple system atrophy (e.g., olivopontocerebellar atrophy,striatonigral degeneration and Shy-Drager syndrome). Therefore, thepresent methods can be administered prophylactically to individuals whohave a known genetic risk of a LBD. Such individuals include thosehaving relatives who have experienced this disease, and those whose riskis determined by analysis of genetic or biochemical markers. Geneticmarkers of risk toward PD include mutations in the synuclein or Parkin,UCHLI, and CYP2D6 genes; particularly mutations at position 53 of thesynuclein gene. Individuals presently suffering from Parkinson's diseasecan be recognized from its clinical manifestations including restingtremor, muscular rigidity, bradykinesia and postural instability.

In some methods, is free of clinical symptoms, signs and/or risk factorsof any amyloidogenic disease and suffers from at least onesynucleinopathic disease. In some methods, the patient is free ofclinical symptoms, signs and/or risk factors of any diseasecharacterized by extracellular amyloid deposits. In some methods, thepatient is free of diseases characterized by amyloid deposits of Aβpeptide. In some methods, the patient is free of clinical symptoms,signs and/or risk factors of Alzheimer's disease. In some methods, thepatient is free of clinical symptoms, signs and/or risk factors ofAlzheimer's disease, cognitive impairment, mild cognitive impairment andDown's syndrome. In some methods, the patient has concurrent Alzheimer'sdisease and a disease characterized by Lewy bodies. In some methods, thepatient has concurrent Alzheimer's disease and a disease characterizedsynuclein accumulation. In some methods, the patient has concurrentAlzheimer's and Parkinson's disease.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,or 30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60, or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent (e.g., alpha-SN peptide or Aβ, or both) over time. If the responsefalls, a booster dosage is indicated.

Optionally, presence of absence of symptoms, signs or risk factors of adisease is determined before beginning treatment.

VI. Patients Amenable to Anti-Amyloid Component Treatment Regimes

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showing symptomsof amyloidosis. In the case of Alzheimer's disease, virtually anyone isat risk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who do have a known genetic risk ofAlzheimer's disease or any of the other hereditary amyloid diseases.Such individuals include those having relatives who have experiencedthis disease, and those whose risk is determined by analysis of geneticor biochemical markers. Genetic markers of risk toward Alzheimer'sdisease include mutations in the APP gene, particularly mutations atposition 717 and positions 670 and 671 referred to as the Hardy andSwedish mutations respectively (see Hardy, TINS, supra). Other markersof risk are mutations in the presenilin genes, PS1 and PS2, and ApoE4,family history of AD, hypercholesterolemia or atherosclerosis.Individuals presently suffering from Alzheimer's disease can berecognized from characteristic dementia, as well as the presence of riskfactors described above. In addition, a number of diagnostic tests areavailable for identifying individuals who have AD. These includemeasurement of CSF tau and Aβ42 levels. Elevated tau and decreased Aβ42levels signify the presence of AD. Individuals suffering fromAlzheimer's disease can also be diagnosed by MMSE or ADRDA criteria asdiscussed in the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent (e.g., NAC) over time, along the lines described in VII Methods ofMonitoring and Diagnosis, below. If the response falls, a booster dosageis indicated.

VII. Treatment Regimes

In general treatment regimes involve administering an agent effective toinduce an immunogenic response to alpha-SN and/or an agent effective toinduce an immunogenic response to Aβ to a patient. In prophylacticapplications, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk of a LBDor another synucleopathic disease in a regime comprising an amount andfrequency of administration of the composition or medicament sufficientto eliminate or reduce the risk, lessen the severity, or delay theoutset of the disease, including physiological, biochemical, histologicand/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presenting during development ofthe disease. In therapeutic applications, compositions or medicates areadministered to a patient suspected of, or already suffering from such adisease in a regime comprising an amount and frequency of administrationof the composition sufficient to cure, or at least partially arrest, thesymptoms of the disease (physiological, biochemical, histologic and/orbehavioral), including its complications and intermediate pathologicalphenotypes in development of the disease. For example, in some methodstreatment effects at least partial clearance of Lewy bodies, at leastpartial disaggregation of Lewy bodies and/or reduces levels ofalpha-synuclein oligomers in synapses. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. A combination of amount and dosagefrequency adequate to accomplish therapeutic or prophylactic treatmentis defined as a therapeutically or prophylatically-effective regime. Inboth prophylactic and therapeutic regimes, agents are usuallyadministered in several dosages until a sufficient immune response hasbeen achieved. Typically, the immune response is monitored and repeateddosages are given if the immune response starts to wane.

In some methods, administration of an agent results in reduction ofintracellular levels of aggregated synuclein. In some methods,administration of an agent results in improvement in a clinical symptomof a LBD, such as motor function in the case of Parkinson's disease. Insome methods, reduction in intracellular levels of aggregated synucleinor improvement in a clinical symptom of disease is monitored atintervals after administration of an agent.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnonhuman mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.The amount of immunogen depends on whether adjuvant is alsoadministered, with higher dosages being required in the absence ofadjuvant. The amount of an immunogen for administration sometimes variesfrom 1-500 μg per patient and more usually from 5-500 μg per injectionfor human administration. Occasionally, a higher dose of 1-2 mg perinjection is used. Typically about 10, 20, 50 or 100 μg is used for eachhuman injection. The mass of immunogen also depends on the mass ratio ofimmunogenic epitope within the immunogen to the mass of immunogen as awhole. Typically, 10⁻³ to 10⁻⁵ micromoles of immunogenic epitope areused for microgram of immunogen. The timing of injections can varysignificantly from once a day, to once a year, to once a decade. On anygiven day that a dosage of immunogen is given, the dosage is greaterthan 1 μg/patient and usually greater than 10 μg/patient if adjuvant isalso administered, and greater than 10 μg/patient and usually greaterthan 100 μg/patient in the absence of adjuvant. A typical regimenconsists of an immunization followed by booster injections at timeintervals, such as 6 week intervals. Another regimen consists of animmunization followed by booster injections 1, 2 and 12 months later.Another regimen entails an injection every two months for life.Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg or, in other words, 70 mgs or700 mgs or within the range of 70-700 mgs, respectively, for a 70 kgpatient. An exemplary treatment regime entails administration once perevery two weeks or once a month or once every 3 to 6 months. In somemethods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toalpha-SN in the patient. In some methods, dosage is adjusted to achievea plasma antibody concentration of 1-1000 ug/ml and in some methods25-300 ug/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. In general, human antibodies show the longesthalf life, followed by humanized antibodies, chimeric antibodies, andnonhuman antibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patent canbe administered a prophylactic regime.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intrathecal, intraperitoneal, intranasal or intramuscularmeans for prophylactic and/or therapeutic treatment. The most typicalroute of administration of an immunogenic agent is subcutaneous althoughother routes can be equally effective. The next most common route isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. In some methods, agents areinjected directly into a particular tissue where deposits haveaccumulated, for example intracranial injection. Intramuscular injectionor intravenous infusion are preferred for administration of antibody. Insome methods, particular therapeutic antibodies are injected directlyinto the cranium. In some methods, antibodies are administered as asustained release composition or device, such as a Medipad™ device.

As noted above, agents inducing an immunogenic response against alpha-SNand Aβ respectively can be administered in combination. The agents canbe combined in a single preparation or kit for simultaneous, sequentialor separate use. The agents can occupy separate vials in the preparationor kit or can be combined in a single vial. These agents of theinvention can optionally be administered in combination with otheragents that are at least partly effective in treatment of LBD. In thecase of Parkinson's Disease and Down's syndrome, in which LBs occur inthe brain, agents of the invention can also be administered inconjunction with other agents that increase passage of the agents of theinvention across the blood-brain barrier.

Immunogenic agents of the invention, such as peptides, are sometimesadministered in combination with an adjuvant. A variety of adjuvants canbe used in combination with a peptide, such as alpha-SN, to elicit animmune response. Preferred adjuvants augment the intrinsic response toan immunogen without causing conformational changes in the immunogenthat affect the qualitative form of the response. Preferred adjuvantsinclude aluminum hydroxide and aluminum phosphate, 3 De-O-acylatedmonophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem ResearchInc., Hamilton, Mont., now part of Corixa). Stimulon™ QS-21 is atriterpene glycoside or saponin isolated from the bark of the Quillaj aSaponaria Molina tree found in South America (see Kensil et al., inVaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman,Plenum Press, N Y, 1995); U.S. Pat. No. 5,057,540), (AquilaBioPharmaceuticals, Framingham, Mass.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killedmycobacteria. Another adjuvant is CpG (WO 98/40100). Alternatively,alpha-SN or Aβ can be coupled to an adjuvant. However, such couplingshould not substantially change the conformation of alpha-SN so as toaffect the nature of the immune response thereto. Adjuvants can beadministered as a component of a therapeutic composition with an activeagent or can be administered separately, before, concurrently with, orafter administration of the therapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such as alumhydroxide, alum phosphate, alum sulfate. Such adjuvants can be used withor without other specific immunostimulating agents such as MPL or 3-DMP,QS-21, polymeric or monomeric amino acids such as polyglutamic acid orpolylysine. Another class of adjuvants is oil-in-water emulsionformulations. Such adjuvants can be used with or without other specificimmunostimulating agents such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi ImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™).

Another class of preferred adjuvants is saponin adjuvants, such asStimulon™ (QS-21, Aquila, Framingham, Mass.) or particles generatedtherefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX.Other adjuvants include RC-529, GM-CSF and Complete Freund's Adjuvant(CFA) and Incomplete Freund's Adjuvant (IFA). Other adjuvants includecytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-6, IL-12,IL13, and IL-15), macrophage colony stimulating factor (M-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), and tumornecrosis factor (TNF). Another class of adjuvants is glycolipidanalogues including N-glycosylamides, N-glycosylureas andN-glycosylcarbamates, each of which is substituted in the sugar residueby an amino acid, as immuno-modulators or adjuvants (see U.S. Pat. No.4,855,283). Heat shock proteins, e.g., HSP70 and HSP90, may also be usedas adjuvants.

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the immunogenic formulationcontaining the adjuvant, the route of administration, the dosingschedule, the efficacy of the adjuvant for the species being vaccinated,and, in humans, a pharmaceutically acceptable adjuvant is one that hasbeen approved or is approvable for human administration by pertinentregulatory bodies. For example, Complete Freund's adjuvant is notsuitable for human administration. Alum, MPL and QS-21 are preferred.Optionally, two or more different adjuvants can be used simultaneously.Preferred combinations include alum with MPL, alum with QS-21, MPL withQS-21, MPL or RC-529 with GM-CSF, and alum, QS-21 and MPL together.Also, Incomplete Freund's adjuvant can be used (Chang et al., AdvancedDrug Delivery Reviews 32, 173-186 (1998)), optionally in combinationwith any of alum, QS-21, and MPL and all combinations thereof.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). Thus, any agent (e.g., fragment of alpha synuclein or antibodyspecifically binding to alpha synuclein) can be used in the manufactureof a medicament for treatment of synucleinopathic disease. The preferredform depends on the intended mode of administration and therapeuticapplication. The compositions can also include, depending on theformulation desired, pharmaceutically-acceptable, non-toxic carriers ordiluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl. Compositions for parenteral administration aretypically substantially sterile, substantially isotonic and manufacturedunder GMP conditions of the FDA or similar body. For example,compositions containing biologics are typically sterilized by filtersterilization. Compositions can be formulated for single doseadministration.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. Compositions can be formulated in unit dosage form (i.e.,the formulation contains sufficient of the active ingredient for onedosage to one patient).

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995);Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).

VIII. Methods of Monitoring and Methods of Diagnosis

The invention provides methods of detecting an immune response againstalpha-SN peptide and/or Aβ peptide in a patient suffering from orsusceptible to a LBD. The methods are particularly useful for monitoringa course of treatment being administered to a patient. The methods canbe used to monitor both therapeutic treatment on symptomatic patientsand prophylactic treatment on asymptomatic patients. The methods areuseful for monitoring both active immunization (e.g., antibody producedin response to administration of immunogen) and passive immunization(e.g., measuring level of administered antibody).

1. Active Immunization

Some methods entail determining a baseline value of an immune responsein a patient before administering a dosage of agent, and comparing thiswith a value for the immune response after treatment. A significantincrease (i.e., greater than the typical margin of experimental error inrepeat measurements of the same sample, expressed as one standarddeviation from the mean of such measurements) in value of the immuneresponse signals a positive treatment outcome (i.e., that administrationof the agent has achieved or augmented an immune response). If the valuefor immune response does not change significantly, or decreases, anegative treatment outcome is indicated. In general, patients undergoingan initial course of treatment with an immunogenic agent are expected toshow an increase in immune response with successive dosages, whicheventually reaches a plateau. Administration of agent is generallycontinued while the immune response is increasing. Attainment of theplateau is an indicator that the administered of treatment can bediscontinued or reduced in dosage or frequency.

In other methods, a control value (i.e., a mean and standard deviation)of immune response is determined for a control population. Typically theindividuals in the control population have not received prior treatment.Measured values of immune response in a patient after administering atherapeutic agent are then compared with the control value. Asignificant increase relative to the control value (e.g., greater thanone standard deviation from the mean) signals a positive treatmentoutcome. A lack of significant increase or a decrease signals a negativetreatment outcome. Administration of agent is generally continued whilethe immune response is increasing relative to the control value. Asbefore, attainment of a plateau relative to control values in anindicator that the administration of treatment can be discontinued orreduced in dosage or frequency.

In other methods, a control value of immune response (e.g., a mean andstandard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose immune responses have reached a plateau in response to treatment.Measured values of immune response in a patient are compared with thecontrol value. If the measured level in a patient is not significantlydifferent (e.g., more than one standard deviation) from the controlvalue, treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment regime, for example, use of adifferent adjuvant may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for immuneresponse to determine whether a resumption of treatment is required. Themeasured value of immune response in the patient can be compared with avalue of immune response previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same sample) is an indication that treatmentcan be resumed. Alternatively, the value measured in a patient can becompared with a control value (mean plus standard deviation) determinedin a population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousor cerebrospinal fluid from the patient. The sample is analyzed forindication of an immune response to any form of alpha-SN, typically NAC,or Aβ. The immune response can be determined from the presence of, e.g.,antibodies or T-cells that specifically bind to alpha-SN or AB. ELISAmethods of detecting antibodies specific to alpha-SN are described inthe Examples section. Methods of detecting reactive T-cells have beendescribed above (see Definitions). In some methods, the immune responseis determined using a clearing assay, such as described in Section IIIabove. In such methods, a tissue or blood sample from a patient beingtested is contacted with LBs (e.g., from a synuclein/hAPP transgenicmouse) and phagocytic cells bearing Fc receptors. Subsequent clearing ofthe LBs is then monitored. The existence and extent of clearing responseprovides an indication of the existence and level of antibodieseffective to clear alpha-SN in the tissue sample of the patient undertest.

2. Passive Immunization

In general, the procedures for monitoring passive immunization aresimilar to those for monitoring active immunization described above.However, the antibody profile following passive immunization typicallyshows an immediate peak in antibody concentration followed by anexponential decay. Without a further dosage, the decay approachespretreatment levels within a period of days to months depending on thehalf-life of the antibody administered. For example the half-life ofsome human antibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to alpha-SN in thepatient is made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

3. Diagnostic Kits

The invention further provides diagnostic kits for performing thediagnostic methods described above. Typically, such kits contain anagent that specifically binds to antibodies to alpha-SN. The kit canalso include a label. For detection of antibodies to alpha-SN, the labelis typically in the form of labeled anti-idiotypic antibodies. Fordetection of antibodies, the agent can be supplied prebound to a solidphase, such as to the wells of a microtiter dish. Kits also typicallycontain labeling providing directions for use of the kit. The labelingmay also include a chart or other correspondence regime correlatinglevels of measured label with levels of antibodies to alpha-SN. The termlabeling refers to any written or recorded material that is attached to,or otherwise accompanies a kit at any time during its manufacture,transport, sale or use. For example, the term labeling encompassesadvertising leaflets and brochures, packaging materials, instructions,audio or video cassettes, computer discs, as well as writing imprinteddirectly on kits.

The invention also provides diagnostic kits for performing in vivoimaging. Such kits typically contain an antibody binding to an epitopeof alpha-SN, e.g., within NAC. Preferably, the antibody is labeled or asecondary labeling reagent is included in the kit. Preferably, the kitis labeled with instructions for performing an in vivo imaging assay.

In one embodiment the antibody is selected from mAb 6H7, mAb 8A5, mAb9E4, mAb 1H7, or mAb 11A5 or a binding fragment thereof. Theaforementioned anti-alpha synuclein antibodies also may be used inassays as described in U.S. patent publication No. 2005196818, theentire content of which is incorporated herein by reference.

IX. In Vivo Imaging

The invention provides methods of in vivo imaging LBs in a patient. Suchmethods are useful to diagnose or confirm diagnosis of PD, or otherdisease associated with the presence of LBs in the brain, orsusceptibility thereto. For example, the methods can be used on apatient presenting with symptoms of dementia. If the patient has LBs,then the patient is likely suffering from, e.g. PD. The methods can alsobe used on asymptomatic patients. Presence of abnormal deposits ofamyloid indicates susceptibility to future symptomatic disease. Themethods are also useful for monitoring disease progression and/orresponse to treatment in patients who have been previously diagnosedwith Parkinson's disease.

The methods work by administering a reagent, such as antibody that bindsto alpha-SN in the patient and then detecting the agent after it hasbound. Preferred antibodies bind to alpha-SN deposits in a patientwithout binding to full length NACP polypeptide. Antibodies binding toan epitope of alpha-SN within NAC are particularly preferred. Ifdesired, the clearing response can be avoided by using antibodyfragments lacking a full length constant region, such as Fabs. In somemethods, the same antibody can serve as both a treatment and diagnosticreagent. In general, antibodies binding to epitopes N-terminal ofalpha-SN do not show as strong signal as antibodies binding to epitopesC-terminal, presumably because the N-terminal epitopes are inaccessiblein LBs (Spillantini et al PNAS, 1998). Accordingly, such antibodies areless preferred.

Diagnostic reagents can be administered by intravenous injection intothe body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labeled, although in some methods, the primary reagentwith affinity for alpha-SN is unlabelled and a secondary labeling agentis used to bind to the primary reagent. The choice of label depends onthe means of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size and/or intensity oflabeled loci to corresponding base line values. The base line values canrepresent the mean levels in a population of undiseased individuals.Base line values can also represent previous levels determined in thesame patient. For example, base line values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the base line values. A decrease in values relative tobase line signals a positive response to treatment.

EXAMPLES Example I. Immunization of Human Alpha-Synuclein TransgenicMice with Human Alpha-Synuclein Results in the Production of High TiterAnti-Alpha-Synuclein Antibodies that Cross the Blood-Brain Barrier

Full-length recombinant human alpha-SN was resuspended at aconcentration of 1 mg/ml in 1× phosphate buffered saline (PBS). For eachinjection, 50 μl of alpha-SN was used; giving a final concentration of50 μg per injection to which 150 μl of 1×PBS was added. CompleteFreund's adjuvant (CFA) was then added 1:1 to either alpha-SN or PBSalone (control), vortexed and sonicated to completely resuspend theemulsion. For the initial injections, eight D line human alpha-SNtransgenic (tg) single transgenic 4-7 months old mice (Masliah, et al.Science 287:1265-1269 (2000) received injections of human alpha-SN inCFA and, as control, four D line human alpha-SN tg mice receivedinjections of PBS in CFA. Mice received a total of 6 injections. Threeinjections were performed at two weeks intervals and then 3 injectionsat one month intervals. Animals were sacrificed using NIH Guidelines forthe humane treatment of animals 5 months after initiation of theexperiment. After blood samples were collected for determination ofantibody titers, brains were immersion-fixed for 4 days in 4%paraformaldehyde in PBS. Levels of antibodies against human alpha-SN byELISA are shown in Table 1. The treated mice are divided into two groupsby titer. The first group developed a moderate titer of 2-8,000. Thesecond group developed a high titer of 12000-30000. No titer was foundin control mice. Neuropathological analysis showed that mice producinghigh titers had a marked decrease in the size of synuclein incusions.Mice producing moderate titers showed a smaller decrease. FIG. 2 (panelsa-d) show synuclein inclusions in (a) a nontransgenic mouse, (b) atransgenic mouse treated with CFA only, (c) a transgenic mouse immunizedwith alpha synuclein and CFA that developed a moderate titer and (d) atransgenic mouse immunized with alpha synuclein and CFA that developed ahigher titer. Samples were visualized by immunostaining with ananti-human alpha-SN antibody. FIG. 2 shows synuclein inclusions in panel(b) but not panel (a). In panel (c), treated mouse, moderate titers, theinclusions are somewhat reduced in intensity. In panel (d) theinclusions are markedly reduced in intensity. Panels (e)-(h) show levelsof anti-IgG in the brains same four mice as panels (a) to (d)respectively. It can be seen that IgG is present in panels (g) and to agreater extent in panel (h). The data shows that peripherallyadministered antibodies to alpha-SN cross the blood brain barrier andreach the brain. Panels (i) to (1) showing staining for GAP, a marker ofastroglial cells, again for the same four mice as in the first two rowsof the figure. It can be seen that panels (k) and (1) show moderatelyincreased staining compared with (i) and (j). These data show thatclearing of synuclein deposits is accompanied by a mild astroglial andmicroglial reaction.

TABLE 1 Syn (+) Geno- Age at Treatment/ inclusions/ Group type n= SacLength Titers mm2 I Syn 4 10-13 a-syn + CFA 2,000- 15-29 Tg mo 50 ug/injfor 3 mo 8,000 sac'd 3 mo later II Syn 4 10-13 a-syn + CFA 12,000- 10-22Tg mo 50 ug/inj for 3 mo 30,000 sac'd 3 mo later III Syn 4 10-13 PBS +CFA for 0 18-29 Tg mo 3 mo sac'd 3 mo later

Example II. In Vitro Screen for Antibodies Clearing Synuclein Inclusions

GT1-7 neuronal cell (Hsue et al. Am. J. Pathol. 157:401-410 (2000)) weretransfected with a pCR3.1-T expression vector (Invitrogen, Carlsbad,Calif.) expressing murine alpha-SN and compared with cells transfectedwith expression vector alone (FIGS. 3, B and A respectively). Cellstransfected with vector alone (A) have a fibroblastic appearance whilecells transfected with alpha-SN are rounded, with inclusion bodies atthe cell surface visible via both light and confocal scanningmicroscopy. Transfected cells were then treated with rabbit preimmuneserum (FIG. 3 C) or 67-10, an affinity purified rabbit polyclonalantibody against a murine alpha-SN C terminal residues 131-140 (Iwai, etal., Neuron 14:467 (1995) (FIG. 3D). It can be seen that the inclusionbodies stain less strongly in panel D than in panel C indicating thatthe antibody against alpha synuclein was effective in clearing orpreventing the development of inclusions. FIG. 4 shows a gel analysis ofparticulate and cytosolic fractions of GT1-7 transfected cells treatedwith the rabbit preimmune serum and 67-10 polyclonal antibody. It can beseen that the synuclein levels in the cytosolic fraction is largelyunchanged by treatment with preimmune serum or antibody to alpha-SN.However, the alpha-SN band disappears in the membrane fraction of GT1-7cells treated with antibody to alpha-SN. These data indicates that thealpha synuclein antibody activity results in the clearance of synucleinassociated with the cellular membrane.

Transfected GT1-7 cells can be used to screen antibodies for activity inclearing synuclein incusions with detection either byimmunohistochemical analysis, light microscopy as in FIG. 3 or by gelanalysis as in FIG. 4.

Example III. Prophylactic and Therapeutic Efficacy of Immunization withAlpha-Synuclein

i. Immunization of Human Alpha-Synuclein Tg Mice

For this study, heterozygous human alpha-SN transgenic (tg) mice (LineD) (Masliah et al., 2000, Science 286:1265-69) and nontransgenic (nontg)controls are used. Experimental animals are divided into 3 groups. Forgroup I, the preventive effects of early immunization by immunizing micefor 8 months beginning at 2 months of age are tested. For group II,young adult mice are vaccinated for 8 months beginning at the age of 6months to determine whether immunization can reduce disease progressiononce moderate pathology had been established. For group III, older miceare immunized for 4 months beginning at the age of 12 months todetermine whether immunization can reduce the severity of symptoms oncerobust pathology has been established. For all groups, mice areimmunized with either recombinant human alpha-SN plus CFA or CFA alone,and for each experiment 20 tg and 10 nontg mice are used. Of them, 10 tgmice are immunized with human alpha-SN+CFA and other 10 tg with CFAalone. Similarly, 5 nontg mice are immunized with human alpha-SN+CFA andthe other 5 with CFA alone. Briefly, the immunization protocol consistsof an initial injection with purified recombinant human alpha-SN (2mg/ml) in CFA, followed by a reinjection 1 month later with humanalpha-SN in combination with IFA. Mice are then re-injected with thismixture once a month. In a small subset of human alpha-SN tg (n=3/each;6-months-old) and nontg (n=3/each; 6-month-old) mice, additionalexperiments consisting of immunization with murine (m) alpha-SN, humanbeta synuclein or mutant (A53T) human alpha-SN are performed.

Levels of alpha-SN antibody are determined using 96-well microtiterplates coated with 0.4m per well of purified full-length alpha-SN byovernight incubation at 4° C. in sodium carbonate buffer, pH 9.6. Wellsare washed 4× with 200 μL each PBS containing 0.1% Tween and blocked for1 hour in PBS-1% BSA at 37° C. Serum samples are serially diluted“in-well”, 1:3, starting in row A, ranging from a 1:150 to 1:328,050dilution. For control experiments, a sample of mouse monoclonal antibodyis run against alpha-SN, no protein, and buffer-only blanks. The samplesare incubated overnight at 4° C. followed by a 2-hour incubation withgoat anti-mouse IgG alkaline phosphatase-conjugated antibody (1:7500,Promega, Madison, Wis.). Atto-phos® alkaline phophatase fluorescentsubstrate is then added for 30 minutes at room temperature. The plate isread at an excitation wavelength of 450 nm and an emission wavelength of550 nm. Results are plotted on a semi-log graph with relativefluorescence units on the ordinate and serum dilution on the abscissa.Antibody titer is defined as the dilution at which there was a 50%reduction from maximal antibody binding.

For each group, at the end of the treatment, mice undergo motorassessment in the rotarod, as described (Masliah, et al. (2000)). Afteranalysis, mice are euthanized and brains are removed for detailedneurochemical and neuropathological analysis as described below.Briefly, the right hemibrain is frozen and homogenized fordeterminations of aggregated and non-aggregated human alpha-SNimmunoreactivity by Western blot (Masliah, et al. (2000)). The lefthemibrain is fixed in 4% paraformaldehyde, serially sectioned in thevibratome for immunocytochemistry and ultrastructural analysis.

ii. Immunocytochemical and Neuropathological Analysis.

In order to determine if immunization decreases, human alpha-SNaggregation sections are immunostained with a rabbit polyclonal antibodyagainst human alpha-SN (1:500). After an overnight incubation at 4° C.,sections are incubated with biotinylated anti-rabbit secondary antibodyfollowed by Avidin D-Horseradish peroxidase (HRP) complex (1:200, ABCElite, Vector). Sections are also immunostained with biotinylatedanti-rabbit, mouse or human secondary alone. The experiments with theanti-mouse secondary determine whether the antibodies against humanalpha-SN cross into the brain. The reaction is visualized with 0.1%3,3-diaminobenzidine tetrahydrochloride (DAB) in 50 mM Tris-HCl (pH 7.4)with 0.001% H₂O₂ and sections are then mounted on slided under Entellan.Levels of immunoreactivity are semiquantitatively assessed by opticaldensitometry using the Quantimet 570C. These sections are also studiedby image analysis to determine the numbers of alpha-SN immunoractiveinclusions and this reliable measure of alpha-SN aggregation acts as avaluable index of the anti-aggregation effects of vaccination (Masliah,et al. (2000)).

Analysis of patterns of neurodegeneration is achieved by analyzingsynaptic and dendritic densities in the hippocampus, frontal cortex,temporal cortex and basal ganglia utilizing vibratome sectionsdouble-immunolabeled for synaptophysin and microtubule-associatedprotein 2 (MAP2) and visualized with LSCM. Additional analysis ofneurodegeneration is achieved by determining tyrosine hydroxylase (TH)immunoreactivity in the caudoputamen and substantia nigra (SN) aspreviously described (Masliah, et al. (2000)). Sections will be imagedwith the LSCM and each individual image is interactively thresholdedsuch that the TH-immunoreactive terminals displaying pixel intensitywithin a linear range are included. A scale is set to determine thepixel to μm ratio. Then, this information is used to calculate the %area of the neuropil covered by TH-immunoractive terminals. These samesections are also utilized to evaluate the numbers of TH neurons in theSN.

To assess the patterns of immune response to immunization,immunocytochemical and ultrastructural analysis with antibodies againsthuman GFAP, MCH class II, Mac 1, TNF-alpha, ILlbeta and IL6 areperformed in the brain sections of nontg and alpha-SN tg mice immunizedwith recombinant human alpha-SN and control immunogens.

iii. Behavioral Analysis.

For locomotor activity mice are analyzed for 2 days in the rotarod (SanDiego) Instruments, San Diego, Calif.), as previously described(Masliah, et al. (2000)). On the first day mice are trained for 5trials: the first one at 10 rpm, the second at 20 rpm and the third tofifth at 40 rpm. On the second day, mice are tested for 7 trials at 40rpm each. Mice are placed individually on the cylinder and the speed ofrotation is increased from 0 to 40 rpm over a period of 240 sec. Thelength of time mice remain on the rod (fall Latency) is recorded andused as a measure of motor function.

Example IV. Immunization with Alpha-Synuclein Fragments

Human alpha-SN transgenic mice 10-13 months of age are immunized with 9different regions of alpha-SN to determine which epitopes convey theefficacious response. The 9 different immunogens and one control areinjected i.p. as described above. The immunogens include four humanalpha-SN peptide conjugates, all coupled to sheep anti-mouse IgG via acystine link. Alpha-SN and PBS are used as positive and negativecontrols, respectively. Titers are monitored as above and mice areeuthanized at the end of 3-12 months of injections. Histochemistry,alpha-SN levels, and toxicology analysis is determined post mortem.

i. Preparation of Immunogens

Preparation of coupled alpha-SN peptides: H alpha-SN peptide conjugatesare prepared by coupling through an artificial cysteine added to thealpha-SN peptide using the crosslinking reagent sulfo-EMCS. The alpha-SNpeptide derivatives are synthesized with the following final amino acidsequences. In each case, the location of the inserted cysteine residueis indicated by underlining.

alpha-synuclein 60-72 (NAC region) peptide: (SEQ ID NO: 54)NH2-KEQVTNVCGGAVVT-COOH alpha-synuclein 73-84 (NAC region) peptide:(SEQ ID NO: 55) NH2-GVTAVAQKTVECG-COOH alpha-synuclein 102-112 peptide:(SEQ ID NO: 56) NH2-C-amino-heptanoic acid- KNEEGAPCQEG-COOHalpha-synuclein 128-140 peptide: (SEQ ID NO: 57)Ac-NH-PSEEGYQDYEPECA-COOH

To prepare for the coupling reaction, ten mg of sheep anti-mouse IgG(Jackson ImmunoResearch Laboratories) is dialyzed overnight against 10mM sodium borate buffer, pH 8.5. The dialyzed antibody is thenconcentrated to a volume of 2 mL using an Amicon Centriprep tube. Ten mgsulfo-EMCS [N (ε-maleimidocuproyloxy) succinimide] (Molecular SciencesCo.) is dissolved in one mL deionized water. A 40-fold molar excess ofsulfo-EMCS is added drop wise with stirring to the sheep anti-mouse IgGand then the solution is stirred for an additional ten min. Theactivated sheep anti-mouse IgG is purified and buffer exchanged bypassage over a 10 mL gel filtration column (Pierce Presto Column,obtained from Pierce Chemicals) equilibrated with 0.1 M NaPO4, 5 mMEDTA, pH 6.5. Antibody containing fractions, identified by absorbance at280 nm, are pooled and diluted to a concentration of approximately 1mg/mL, using 1.4 mg per OD as the extinction coefficient. A 40-foldmolar excess of alpha-SN peptide is dissolved in 20 mL of 10 mM NaPO4,pH 8.0, with the exception of the alpha-SN peptide for which 10 mg isfirst dissolved in 0.5 mL of DMSO and then diluted to 20 mL with the 10mM NaPO4 buffer. The peptide solutions are each added to 10 mL ofactivated sheep anti-mouse IgG and rocked at room temperature for 4 hr.The resulting conjugates are concentrated to a final volume of less than10 mL using an Amicon Centriprep tube and then dialyzed against PBS tobuffer exchange the buffer and remove free peptide. The conjugates arepassed through 0.22 μm-pore size filters for sterilization and thenaliquoted into fractions of 1 mg and stored frozen at −20° C. Theconcentrations of the conjugates are determined using the BCA proteinassay (Pierce Chemicals) with horse IgG for the standard curve.Conjugation is documented by the molecular weight increase of theconjugated peptides relative to that of the activated sheep anti-mouseIgG.

Example V. Passive Immunization with Antibodies to Alpha-Synuclein

Human alpha-SN mice each are injected with 0.5 mg in PBS ofanti-alpha-SN monoclonals as shown below. All antibody preparations arepurified to have low endotoxin levels. Monoclonals can be preparedagainst a fragment by injecting the fragment or longer form of alpha-SNinto a mouse, preparing hybridomas and screening the hybridomas forantibody that specifically binds to a desired fragment of alpha-SNwithout binding to other nonoverlapping fragments of alpha-SN.

Mice are injected ip as needed over a 4 month period to maintain acirculating antibody concentration measured by ELISA titer of greaterthan 1:1000 defined by ELISA to alpha-SN or other immunogen. Titers aremonitored as above and mice are euthanized at the end of 6 months ofinjections. Histochemistry, alpha-SN levels and toxicology are performedpost mortem.

Example VI. Aβ Immunization of Syn/APP Transgenic Mice

This experiment compares the effects of Aβ immunization on three typesof transgenic mice: transgenic mice with an alpha synuclein transgene(SYN), APP mice with an APP transgene (Games et al.) and doubletransgenic SYN/APP mice produced by crossing the single transgenic. Thedouble transgenic mice are described in Masliah et al., PNAS USA98:12245-12250 (2001). These mice represent a model of individualshaving both Alzheimer's and Parkinson's disease. Table 2 shows thedifferent groups, the age of the mice used in the study, the treatmentprocedure and the titer of antibodies to Aβ. It can be seen that asignificant titer was generated in all three types of mice. FIG. 5 showsthe % area covered by amyloid plaques of Aβ in the brain determined byexamination of brain sections from treated subjects by microscopy.Substantial deposits accumulate in the APP and SYN/APP mice but not inthe SYN mice or nontransgenic controls. The deposits are greater in theSYN/APP double transgenic mice. Immunization with Aβ1-42 reduces thedeposits in both APP and SYN/APP mice. FIG. 6 shows synuclein depositsin the various groups of mice as detected by confocal laser scanning andlight microscopy. Synuclein deposits accumulate in the SYN and SYN/APPmice treated with CFA only. However, in the same types of mice treatedwith Aβ1-42 and CFA there is a marked reduction in the level ofsynuclein deposit. These data indicate that treatment with Aβ iseffective not only in clearing Aβ deposits but also in clearing depositsof synuclein. Therefore, treatment with Aβ or antibodies thereto isuseful in treating not only Alzheimer's disease but combined Alzheimer'sand Parkinson's disease, and Parkinson's disease in patients free ofAlzheimer's disease. The titer of antiAβ antibodies in SYN/APP micecorrelated with decreased formation of synuclein inclusions (r=−0.71,p<0.01).

TABLE 2 Group n = Age Treatment/ Length Ab Titers SYN 4 12-20 mo Ab inj.50 ug/inj 10,000-58,000 for 6 mo SYN 2 12-20 mo Sal inj. for 6 mo 0 APP2 12-20 mo Ab inj. 50 ug/inj 25,000 for 6 mo APP 2 12-20 mo Sal inj. for6 mo 0 SYN/APP 4 12-20 mo Ab inj. 50 ug/inj 1,000-50,000 for 6 moSYN/APP 2 12-20 mo Sal inj. for 6 mo 0

Example VII. Ex Vivo Screening Assay for Activity of an Antibody AgainstAmyloid Deposits

To examine the effect of antibodies on plaque clearance, we establishedan ex vivo assay in which primary microglial cells were cultured withunfixed cryostat sections of either PDAPP mouse or human AD brains.Microglial cells were obtained from the cerebral cortices of neonateDBA/2N mice (1-3 days). The cortices were mechanically dissociated inHMS⁻⁻ (Hanks' Balanced Salt Solution, Sigma) with 50 μg/ml DNase I(Sigma). The dissociated cells were filtered with a 100 μm cell strainer(Falcon), and centrifuged at 1000 rpm for 5 minutes. The pellet wasresuspended in growth medium (high glucose DMEM, 10% FBS, 25 ng/mlrmGM-CSF), and the cells were plated at a density of 2 brains per T-75plastic culture flask. After 7-9 days, the flasks were rotated on anorbital shaker at 200 rpm for 2 h at 37° C. The cell suspension wascentrifuged at 1000 rpm and resuspended in the assay medium.

10-μm cryostat sections of PDAPP mouse or human AD brains (post-morteminterval <3 hr) were thaw mounted onto poly-lysine coated round glasscoverslips and placed in wells of 24-well tissue culture plates. Thecoverslips were washed twice with assay medium consisting of H-SFM(Hybridoma-serum free medium, Gibco BRL) with 1% FBS, glutamine,penicillin/streptomycin, and 5 ng/ml rmGM-CSF (R&D). Control or anti-ABantibodies were added at a 2× concentration (5 μg/ml final) for 1 hour.The microglial cells were then seeded at a density of 0.8×10⁶ cells/mlassay medium. The cultures were maintained in a humidified incubator(37° C., 5% CO₂) for 24 hr or more. At the end of the incubation, thecultures were fixed with 4% paraformaldehyde and permeabilized with 0.1%Triton-X100. The sections were stained with biotinylated 3D6 followed bya streptavidin/Cy3 conjugate (Jackson ImmunoResearch). The exogenousmicroglial cells were visualized by a nuclear stain (DAPI). The cultureswere observed with an inverted fluorescent microscope (Nikon, TE300) andphotomicrographs were taken with a SPOT digital camera using SPOTsoftware (Diagnostic instruments). For Western blot analysis, thecultures were extracted in 8M urea, diluted 1:1 in reducing tricinesample buffer and loaded onto a 16% tricine gel (Novex). After transferonto immobilon, blots were exposed to 5 μg/ml of the pabAB42 followed byan HRP-conjugated anti-mouse antibody, and developed with ECL (Amersham)

When the assay was performed with PDAPP brain sections in the presenceof an antibody against a NAC marked reduction in the number and size ofplaques indicative of clearing activity of the antibody was observed. Anantibody to NAC was contacted with a brain tissue sample containingamyloid plaques and microglial cells, as discussed above. Rabbit serumwas used as a control.

The same assay was performed with PDAPP brain sections in the presenceseveral antibodies against AB. The ability of the antibodies to inducephagocytosis in the ex vivo assay and to reduce in vivo plaque burden inpassive transfer studies was compared. These results show that efficacyin vivo is due to direct antibody mediated clearance of the plaqueswithin the CNS, and that the ex vivo assay is predictive of in vivoefficacy. (See Tables 16 and 17 of Example XIV of WO 00/72880; and,Example XIV, Table 16, of WO 0072876, both of which are incorporated byreference herein for all purposes.)

Example VIII: Active Immunization with Alpha-Synuclein

A. Materials and Methods

Vaccination of hα-synuclein tg mice. For this study, heterozygous tgmice (Line D) expressing hα-synuclein under the regulatory control ofthe platelet-derived growth factor-β (PDGFβ) promoter (Masliah, 2000,Science 287:1265-69) were used. These animals were selected because theydevelop hα-synuclein immunoreactive inclusions in the brain as well asneurodegenerative and motor deficits that mimic certain aspects of LBD.Experimental animals were divided into two groups. For the first group,a total of 20 young (3 months old) tg mice were immunized for 8 monthswith recombinant hα-synuclein (n=10) or adjuvant alone (n=10). For thesecond group, a total of 20 young adult (6 months old) tg mice wereimmunized for 8 months with recombinant hα-synuclein (n=10) or adjuvantalone (n=10). The immunization protocol consisted first of an injectionwith recombinant ha-synuclein (80 μg/ml; 100 μl) with complete Freund'sadjuvant (CFA). Two weeks later mice received another injection ofhα-synuclein (80 μg/ml; 100 μl) with incomplete FA, followed byre-injection once a month (for the subsequent 7 months) withhα-synuclein (80 μg/ml; 100 μl) in phosphate-buffered saline.Recombinant hα-synuclein was prepared and purified as described inMasliah et al., 2005, Neuron 46:857-68, and tested for endotoxins.

Determination of antibody titers and relative affinity to hα-synuclein.hα-Synuclein antibody levels in plasma were determined using 96-wellmicrotiter plates coated with 0.4 μg per well of purified full-lengthα-synuclein. The samples were incubated overnight at 4° C. followed bywashing and incubation with goat anti-mouse IgG alkaline phosphataseconjugated antibody, (1:7500, Promega, Madison, Wis.). The plate wasread at an excitation wavelength of 450 nm and an emission wavelength of550 nm. Results were plotted on a semi-log graph with relativefluorescence units on the ordinate and serum dilution on the abscissa.Antibody titer was defined as the dilution at which there was a 50%reduction from the maximal antibody binding.

To determine the relative affinity for hα-synuclein by the antibodiesgenerated in the vaccinated mice, two sets of experiments wereperformed. In the first, brain homogenates from non-immunizedhα-synuclein tg mice were run in a minigel, multichannel apparatus(Invitrogen, Carlsbad, Calif.). Each channel was incubated with thediluted serum from each of the mice, blotted onto nitrocellulose andincubated with secondary rabbit anti-mouse antibody followed by I¹²⁵tagged protein A (Alford et al., J. Histochem. Cytochem 42:283-287(1994)). Blots were imaged and analyzed with the PhosphorImager(Molecular Dynamics, Piscataway, N.J.). The immunoreactive band wasquantified using the ImageQuant software (Amersham Biosciences,Piscataway, N.J.). For the second set of experiments, serial vibratomesections from a non-immunized hα-synuclein tg mouse were incubated inthe diluted serum from each of the treated mice followed by biotinylatedhorse anti-mouse IgG (1:100, Vector), Avidin D-horseradish peroxidase(HRP, 1:200, ABC Elite, Vector), and reacted with diaminobenzidinetetrahydrochloride (DAB) containing 0.001% H₂O₂. After microscopicexamination, sections were scored according to the cellular compartmentlabeled (neuronal cell bodies, synapses and inclusions) and the degreeof immunoreactivity (0=none; 1=very mild, 2=mild, 3=moderate,4=intense).

Epitope mapping of hα-synuclein antibodies. The epitopes recognized byha-synuclein antibodies were determined by an ELISA that measures thebinding of an antibody to overlapping linear peptides that covered theentire hα-synuclein sequence. C-terminally biotinylated peptides withsequences of hα-synuclein (Mimotopes, San Diego, Calif.) were preparedas 15 amino acid (aa) long peptides with an overlap of 12 residues and astep of 3 residues per peptide. A total of 43 peptides were used to walkthe entire 140 aa sequence of hα-synuclein with the last peptide havingan overlap of 13 aa and a step of 2 aa. In addition, the last 3 peptideswere repeated, but with the biotinylation occurring on the N-terminal ofthe peptide. This was done to improve the access to the C-terminal ofthe peptides by antibodies and to allow identification of freeC-terminal specific antibodies. Furthermore, other features were addedto this assay to allow a more thorough examination of interactionsbetween antibodies and the non-amyloid β (Aβ) component (NAC) region(61-95) of hα-synuclein. Since the 21^(st) peptide in this assay alreadycontains the free N-terminal of the NAC region, one additionalN-terminally biotinylated peptide that contains the free C-terminal ofthe NAC region was added to complete the assay with a total of 47peptides.

To run the assay, these biotinylated peptides were coated down overnight at 5 nM onto ELISA plates pre-coated with streptavidin (Pierce,Rockford, Ill.). The plates were then washed and serum samples, dilutedto a titer equivalent of 6, were added for a 1-hour incubation. Serumsamples with titers lower than 5,000 were diluted 1:1000 for thisincubation. After another washing step, the bound antibodies weredetected using species-specific second antibodies conjugated to HRP in acolorimetric ELISA format.

Tissue processing. Mice were euthanized and brains removed for detailedneurochemical and neuropathological analysis as described below.Briefly, the right hemibrain was frozen and homogenized fordeterminations of aggregated and unaggregated hα-synucleinimmunoreactivity by Western blot (Masliah et al., 2000, supra). The lefthemibrain was fixed in 4% paraformaldehyde (PFA) and serially sectionedwith the vibratome (Leica, Wetzlar, Germany) for immunocytochemistry(ICC) and ultrastructural analysis.

Synaptosomal preparations and immunoblot analysis. To ascertain theeffects of vaccination on α-synuclein accumulation in the brains of tgmice, synaptosomal fractions were prepared using sucrose gradients andanalyzed by SDS-PAGE on a 10% tris-acetate polyacrylamide gel (NuPAGE™,Invitrogen). Immunoblots were probed with primary antibodies againsthα-synuclein (LB509, 1:1000, Transduction Laboratories, San Diego,Calif.) and synaptophysin (1:20, Chemicon, Temecula, Calif.) andsecondary goat anti-mouse IgG tagged with HRP (1:5000, SantaCruzBiotechnology, Inc., Santa Cruz, Calif.) and visualized by enhancedchemiluminescence and analyzed with a Versadoc XL imaging apparatus(BioRad, Hercules, Calif.).

Neuropathological and immunocytochemical analysis. Briefly, aspreviously described (Masliah et al., 2000), supra, to investigate theeffects of vaccination on ha-synuclein accumulation, serially-sectioned,free-floating, blind-coded vibratome sections were incubated overnightat 4° C. with an affinity purified anti-hasynuclein specific antibody(72-10, rabbit polyclonal, 1:500) prepared as previously described(Masliah et al., 2000, supra) by immunizing rabbits with synthetichα-synuclein peptides consisting of aa 101-124. Incubation with theprimary antibody was followed by biotinylated goat anti-rabbit IgG(1:100, Vector), Avidin D-HRP (1:200, ABC Elite, Vector), and reactedwith DAB tetrahydrochloride containing 0.001% H₂O₂. Sections wereanalyzed with the Quantimet 570C (Leica) in order to determine thenumber of hα-synuclein immunoreactive inclusions in the neocortex. Foreach case, three sections were analyzed and the results were averagedand expressed as numbers per sq mm. Further immunocytochemical analysiswas performed by immunoreacting sections with antibodies against glialmarkers including CD45 (1:1000, DakoCytomation, Carpinteria, Calif.) andglial fibrillary acidic protein (GFAP, 1:500, Chemicon).

Double-immunocytochemical analysis was performed as previously described(Hashimoto et al., Neuron 32:213-223 (2001) to determine the effects ofvaccination on nerve terminal density and hα-synuclein accumulation insynapses. For this purpose, vibratome sections were double-labeled witha polyclonal antibody against hα-synuclein (1:1000) and with themonoclonal antibody against synaptophysin (Chemicon). hα-Synuclein wasdetected with Tyramide Red (1:2000, Roche) and synaptophysin with horseanti-mouse IgG tagged with fluorescein isothiocyanate (FITC). For eachcase, sections were immunolabeled in duplicate and analyzed with thelaser scanning confocal microscope (LSCM) and NIH Image 1.43 software tocalculate the percent area of the neuropil covered bysynaptophysin-immunoreactive terminals in the neocortex (Mucke et al.,J. Neurosci 20:4050-4058 (2000)) and the proportion ofsynaptophysin-immunoreactive terminals that were hα-synuclein positive.In order to confirm the specificity of the primary antibodies, controlexperiments were performed where sections were incubated overnight inthe absence of primary antibody (deleted), with the primary antibodypreadsorbed for 48 hrs with 20-fold excess of the corresponding peptideor with preimmune serum.

All sections were processed simultaneously under the same conditions andexperiments were performed twice in order to assess the reproducibilityof results. Sections were imaged with a Zeiss 63X (N.A. 1.4) objectiveon an Axiovert 35 microscope (Zeiss, Germany) with an attached MRC1024LSCM system (BioRad, Wattford, UK) (Masliah et al., 2000, supra).

Statistical analysis. Statistical comparisons between groups wereperformed utilizing the two-tailed unpaired Student's t-test. Linearregression analysis was performed to ascertain the relationship amongvariables. The Bonferroni correction was applied to account for multiplecomparisons.

B. Results

Characterization of Antibody Titers, Affinity and Epitope Mapping

Antibody titers were analyzed at 3 time points (2 weeks, 6 months and 9months after vaccination) in both experimental groups. Antibody titersvaried considerably among mice, in animals belonging to group I,antibody titers among mice immunized with ha-synuclein ranged from 200to 20,000 (Table 3).

TABLE 3 Summary of α-synuclein titers and immunoblot affinity (correctedfor titer). Anti- Anti- Anti- Anti- Anti- Anti- body body body body bodybody affinity affinity affinity titers titers titers by to to in- (first(second (third Group miniblot synapses clusions bleed) bleed) bleed)Group 109147 ± 1.9 ± 1.2 ± 2332 ± 2772 ± 3644 ± I/α-syn 2700 0.73 0.4500 1176 2365 Group 113 ± 0.4 ± 0 19 ± 30 ± 7 ± I/CFA 113 0.1 6.7 12 4Group 235747 ± 4.1 ± 2.8 ± 3813 ± 2926 ± 1468 ± II/α-syn 74000 0.9 1.01200 976 641 Group 400 ± 0.3 ± 0.1 ± 23 ± 21 ± 0.6 ± II/CFA 358 0.2 0.19 14 0.6In this group the average titers rose slightly over time. Similarly, forgroup II, animals immunized with hα-synuclein showed titers that rangedfrom 200 to 13,000 (Table 3). However, the average titer levels werehigher at the first determination and then decreased over time.Immunoblotting analysis also showed significant variability from mouseto mouse in their ability to recognize hα-synuclein. Overall, levels ofantibody relative affinity was higher in mice from group II compared toimmunized mice from group I (Table 4).

TABLE 4 Summary of correlations between immunoblot affinity,neuropathology and titers. Antibody Antibody Antibody Antibody AntibodyNeuropathological affinity by affinity to affinity to affinity to titers(first markers miniblot synapses inclusions neurons bleed) Number ofα-syn −0.11  0.04 0.12 −0.21  0.1 (+) inclusions % area of neuropil−0.46  −0.41  −0.43  0.06 −0.47  α-syn (+) synapses (p = 0.003) (p =0.009) (p = 0.005) (p = 0.007) % area of neuropil 0.06 0.35 0.01 0.040.12 synaptophysin (+) (p = 0.04) synapses Antibody affinity — 0.74 0.70−0.16  0.85 by miniblot (p = 0.0001) (p = 0.0001) (p = 0.0001) Antibodytiters 0.85 0.62 −0.18  0.81 — (first bleed) (p = 0.0001) (p = 0.0001)(p = 0.0001)By ICC, sera from mice vaccinated with hα-synuclein showed labeling ofneurons, intraneuronal inclusions and presynaptic terminals. Incontrast, mice treated with adjuvant alone showed diffuse andnon-specific mild staining of cell bodies). Sera from mice belonging togroup II showed higher affinity in recognizing hα-synuclein in thesynapses and neurons in the tg mice compared to immunized mice fromgroup I (Table 4).

Epitope mapping studies showed that in mice vaccinated withhα-synuclein, antibodies most frequently recognized peptide epitopeswithin the C-terminus region of hα-synuclein (FIG. 8). In addition,antibodies to additional epitopes were also occasionally recognized. Incontrast, no reactivity or antibody epitopes were detected with the seraof mice treated with CFA alone.

Immunization Reduces hα-Synuclein Accumulation and Preserves SynapticDensity in the Brains of Tg Mice

To determine the effects of immunotherapy on hα-synuclein accumulation,sections were labeled with antibodies against hα-synuclein and analyzedby bright field microscopy or by LSCM. In tg mice, abundant hα-synucleinimmunoreactivity was observed in the neuropil as well as inintraneuronal inclusions. Compared to tg mice treated with CFA alone,mice from both of the immunized groups showed a comparable reduction(approximately 25%) in the number of inclusions in the temporal cortex(FIG. 9A). Moreover, immunization resulted in a decrease in hα-synucleinimmunoreactivity in the neuropil. When compared to tg mice treated withCFA alone, this effect was greater in mice from group II than in micefrom group I (FIG. 9A). To determine if the immunization effects wereindeed related to the antibodies' ability to reduce neuronalhα-synuclein accumulation or to masking effects, control experimentswere performed by comparing the levels of β□ synuclein immunoreactivitybetween CFA alone and hα-synuclein vaccinated tg mice. Consistent withthe known distribution of β□synuclein, a close homologue to α-synuclein(Iwai et al., Neuron 14:467-475 (1994)), abundant β-synucleinimmunoreactivity was observed in the neuropil in association with thepresynaptic terminals and mild immunolabeling was detected in theneuronal cell bodies, but not in the inclusions. Compared to tg micetreated with CFA alone, no differences in the patterns and levels ofβ-synuclein were found in mice immunized with hα-synuclein. To furtherinvestigate the specificity of the effects of the hα-synucleinantibodies, levels of murine (m) α□ synuclein immunoreactivity werecompared between the CFA alone and hα-synuclein vaccinated tg mice.Similar to hα□synuclein, mα□synuclein immunoreactivity was abundant inthe neuropil in association with nerve terminals but was absent in theneuronal cell bodies and in the inclusions. Both in the CFA andhα-synuclein immunized mice, patterns and levels of mα□synuclein werecomparable. Taken together, these studies suggest that vaccinationspecifically affects hα-synuclein but not other related synapticmolecules.

To further ascertain the effects of the immunotherapy on neuropilintegrity, sections were immunostained with an antibody againstsynaptophysin or by electron microscopy. Compared to non-transgenic(nontg) mice, tg mice treated with CFA alone showed an average of 20%decrease in the number of synaptophysin immunolabeled terminals, andlevels of synaptophysin immunoreactivity per synapse remained unchanged(FIG. 9B). In contrast, immunized mice from both groups showed levels ofsynaptophysin immunoreactivity comparable to nontg controls (9B).Further immunocytochemical analysis with antibodies against glialmarkers such as GFAP and CD45 showed a trend toward increasedimmunoreactivity in the brains of tg mice vaccinated with hα-synuclein(FIG. 9C). Consistent with these findings, ultrastructural analysisshowed that in the brains of tg mice immunized with hα-synuclein, theneuropil was well preserved, with intact presynaptic terminals anddendrites and the nerve terminals contained abundant clear vesicles andformed postsynaptic densities. Only occasional electrodense aggregateswere identified in the neuritic processes and overall the mitochondriaand myelin were well preserved.

To better characterize the effects of vaccination on hα-synucleinaggregation in the synapses, double immunocytochemical and Western blotanalysis with synaptosomal preparations was performed. Underphysiological conditions hα-synuclein is localized primarily to thepresynaptic boutons (Iwai et al., 1994, supra) and in LBD and in the tgmice, increased accumulation of hα-synuclein in the synapses isassociated with functional deficits and synapse loss (Hashimoto et al.,2001, supra). To ascertain the effects of vaccination on hα-synucleinaccumulation in the nerve terminals, double immunolabeling studies withantibodies against the presynaptic terminal marker synaptophysin andhα-synuclein and WB analysis with synaptosomal preparations wereperformed. Confocal imaging of double-labeled sections showed that incomparison to hα-synuclein tg mice vaccinated with CFA alone (FIG. 9D),those that were injected with hα-synuclein displayed decreasedaccumulation of hα-synuclein in synaptophysin-immunoreactive nerveterminals in the neocortex (FIG. 9D).

Consistent with the immunocytochemical studies, immunoblot analysisshowed that in the tg mice treated with CFA alone, there were abundanthigher molecular weight bands, possibly reflecting the accumulation ofhα-synuclein immunoreactive inclusions in the synapses (FIG. 10). In theimmunized mice there was a considerable decrease in the accumulation ofhigher molecular weight bands of hα-synuclein and the native band, butno effects were observed on the levels of ma-synuclein. Furthermore,compared to tg mice treated with CFA alone, levels of synaptophysinimmunoreactivity were higher in the synaptosomal preparations fromimmunized mice (FIG. 10). Taken together, these results suggest thatimmunotherapy can ameliorate the neuronal damage in the brains of tgmice by reducing the accumulation of potentially toxic hα-synucleinoligomers in the synapses.

The Effects of Immunization are Dependent on the Relative Affinity ofAntibodies to Recognize Synaptic Terminals

To better understand which factors predict the effectiveness of theimmunotherapy, linear regression analysis was performed between theneuropathological markers of ha-synuclein accumulation and the antibodytiters and affinity. This analysis showed a significant correlationbetween relative antibody affinity by immunoblot and levels ofhα-synuclein immunoreactivity in the synapses but not with the numbersof neuronal inclusions. Similarly, relative antibody affinity torecognize synapses by ICC was inversely correlated with levels ofhα-synuclein in the synapses and directly correlated with the percentarea occupied by synaptophysin-labeled nerve terminals, but not with thenumbers of neuronal inclusions. Levels of antibody reactivity byimmunoblot and ICC were strongly correlated with antibody titers asdetermined by ELISA. Antibody titers were also correlated with thepercent area of the neuropil labeled with the anti-hαsynuclein antibodybut not with the numbers of inclusions in neurons (Table 4). Takentogether, these results suggest that the relative immunoblot reactivityof the anti-human α-synuclein antibodies and to some extent theantibodies' ELISA titers correlate with the reduction of neuronal humanα-synuclein accumulation.

The Anti-Human α-Synuclein Antibodies are Internalized and Bind toSynapses and Inclusion-Containing Neurons in Tg Mice

To determine if trafficking antibodies recognize the characteristicneuronal sites where human α-synuclein accumulates in the brains of tgmice, single and double immunocytochemical analysis was performed withhorse anti-mouse IgG antibodies. These antibodies putatively recognizethe anti-human α-synuclein generated in the immunized animals but not inthe CFA controls. Bright field digital microscopy of the immunolabeledsections showed that in mice immunized with hα-synuclein, thebiotinylated anti-mouse IgG diffusely labeled neuronal cell bodies andneuritic processes in the neuropil. In tg animals treated with CFA alonethere was mild labeling of blood vessels and occasional cells resemblingmicroglia. Double immunostaining experiments confirmed that in thevaccinated mice, neuronal cell bodies labeled by a FITC taggedanti-mouse IgG displayed hα-synuclein immunoreactivity. Compared to tgmice treated with CFA alone, in hα-synuclein vaccinated mice, in someneurons, the anti-mouse IgG and the hα-synuclein immunoreactivity wereco-localized in the periphery of the cell bodies, in other areas the twolabels were detected in the neuritic processes and synapses. Moreover,in several human α-synuclein containing neurons the two markers weredetected in granular subcellular structures averaging in size 0.4-0.8 μmin diameter. Additional double labeling experiments showed that thesegranular structures displayed cathepsin D immunoreactivity, suggestingthat the internalized anti-human a-synuclein antibodies reacted withsynuclein within lysosomes. Consistent with this finding,ultrastructural analysis showed that in some of the neurons of the humanα-synuclein vaccinated mice, electrodense laminated structuressuggestive of lysosomes and phagolysosomes were identified. Takentogether, these results suggest that vaccination with human α-synucleincan promote degradation of this molecule via activation of a lysosomalpathway.

Example IX. Clearance of α-Synuclein Aggregates In Vivo byAdministration of α-Synuclein Antibodies

This example demonstrates clearance of intraneuronal α-synucleinaggregates using monoclonal anti-α-synuclein antibodies that recognizethe α-synuclein termini. The monoclonal antibodies were injected intothe neocortex of transgenic mice that overexpress human α-synuclein andhave intraneuronal α-synucelin aggregates. The two antibodies, onedirected against the N-terminus and the other directed against theC-terminus of α-synuclein, reduced the number of intraneuronalα-synuclein aggregates by up to 80% compared to irrelevant controlantibodies (FIG. 11).

Methods. Monoclonal antibodies recognizing different epitopes of theα-synuclein molecule and irrelevant, isotype-matched control antibodieswere dissolved in sterile phosphate-buffered-saline solution (Table 5)for injection into mice. The animals used were 4 to 8 month-oldheterozygous transgenic mice overexpressing human wildtype α-synucleinin the brain under the transcriptional control of the PDGF promoter.From 4 to 6 different transgenic mice were used for each of theantibodies.

TABLE 5 α-Synuclein Antibodies and Controls Used for IntracerebralInjection in a Transgenic Model of Neuronal Synucleopathy. MonoclonalEpitope/ antibody Specificity Isotype 11A5 α-synuclein IgGl phospoSER1298A5 α-synuclein C-terminus IgGl 9G5 α-synucl ein 91-96 IgGl 23E8α-synuclein 40-55 IgGl 6H7 α-synucl ein N-terminus IgGl 4B1 α-synucleinC-terminus IgG2a 5C12 α-synuclein 109-120 IgG2b 27-1 control IgGlTY11-15 control IgG2a 5B7 control IgG2b

For each mouse, 2 μl of a 2 mg/ml antibody solution were injectedstereotactically under anesthesia into the deep layers of the parietalneocortex of the right brain hemisphere (ipsilateral side). The lefthemispheres (contralateral side) served as an baseline control for eachmouse. Injection sites were sutured and mice were monitored until theyrecovered from anesthesia. The investigator performing the injectionswas blinded as to which antibody was injected each time. Two weeks afterinjection, mice were euthanized following institutional guidelines.Their brains were removed, fixed in 4% paraformaldehyde for 48 h, andcut coronally at 40 μm thickness using a Leica vibratome. Two sectionsper animal (around the injection site) were stained by immunoperoxidasestaining with a polyclonal α-synuclein antibody (ELADW-47, recognizingα-synuclein amino acids 115-122). For each section, intraneuronalα-synuclein aggregates were counted in 4 microscopic fields (20×objective) around the injection site in the ipsilateral hemisphere, andin 4 fields corresponding fields in the contralateral controlhemisphere. The α-synuclein aggregate counts for two sections weresummed for each hemisphere. Finally, for each animal the differencebetween the total α-synuclein aggregate count between the two hemispherewas calculated and expressed as % difference between the contralateraland the ipsilateral hemisphere, thus providing a measure of the effectof α-synuclein antibodies on aggregate clearance for each individualmouse. Sections were blind-coded and the code was broken when analysiswas complete.

The mice can be catagorized into three groups based on which antibodieswere injected:

-   -   Group 1: Mice injected with 11A5, 8A5 or an IgG₁ control.    -   Group 2: Mice injected with 9G5, 23E8, 6H7, or an IgG₁ control.    -   Group 3: Mice injected with 4B1, 5C12, an IgG_(2a) or an        IgG_(2b) control.

Results. The results of the study are shown in FIGS. 11 and 12.Intraneuronal a-synuclein aggregates were cleared by two monoclonalantibodies: 8A5 (also called JH4.8A5) and 6H7 (also called JH17.6H7),both described in PCT patent publication WO 05047860A2 (“Antibodies toAlpha-Synuclein” filed May 26, 2005) and in copending patent applicationSer. No. 10/984,192, both of which are incorporated by reference. MAb6H7 was raised against recombinant human α-synuclein expressed in E.coli and recognizes the amino-terminus of human and mouse α-synucleins.It recognizes an epitope that includes the first three amino acids ofα-synuclein. MAb 6H7 is able to recognize fusion proteins of synucleinin which the tag protein is fused to the N-terminus of synuclein,suggesting a free-amino terminus is not required (though it may bepreferred). MAb 8A5 was raised against purified bovine synucleins(mixture of a and (3) and recognizes an epitope at the carboxy-terminusof human and mouse α-synucleins. MAb 8A5 can bind truncated synucleinterminating at amino acid 139. Preliminary experiments suggest 8A5 has a4-5 fold preference for synuclein with a free C-terminus compared to aC-terminus conjugated to biotin. Both mAb 6H7 and mAb 8A5 also recognizebeta-synuclein. MAb 4B1 recognizes the C-terminal region of synucleinand binds synuclein on western blots, but does not recognize synucleinin solution (i.e., mAb 4B1 does not immunoprecipitate synuclein). FIG.12 shows sections of the contralateral side (left panel; round browndots inside section are α-synuclein aggregates) and ipsilateral side(right panel) of a mouse injected with 8A5. The difference between the8A5-injected mice and the IgG₁-injected controls was statisticallysignificant (p<0.05 by non-parametric Kruskall-Wallis followed by Dunn'spost-hoc test). These results indicate that targeting the α-synucleinC-terminus and/or the N-terminus is therapeutically beneficial insynucleopathies such as PD and DLB. Administration of otheranti-α-synuclein antibodies tested (Table 5, FIG. 11) did not result inclearing of aggregates.

Example X: Clearance of α-Synuclein Aggregates In Vivo by Administrationof α-Synuclein Antibodies

This example demonstrates clearance of intraneuronal α-synucleinaggregates using monoclonal anti-α-synuclein antibodies that recognizethe α-synuclein termini (6H7 and 8A5) as described in Example IX. Thisexample also demonstrates clearance of intraneuronal α-synucleinaggregates using a monoclonal anti-α-synuclein antibody that recognizesan epitope near the C-terminus (9E4). mAb 9E4 recognizes an epitope ofalpha-synuclein in the region of amino acids 118-126.

The monoclonal antibodies were injected 3× in the neocortex of the righthemisphere of transgenic mice that overexpress human α-synuclein andform intraneuronal α-synuclein aggregates. As described above, mAb 6H7,which recognizes the N-terminus of α-synuclein and mAb 8A5, whichrecognizes the C-terminus of α-synuclein, reduced the number ofintraneuronal α-synuclein aggregates by up to 80% compared to anirrelevant control antibody (FIG. 13). In addition, mAb 9E4, whichrecognizes an α-synuclein epitope at aa118-126, had a similarα-synuclein aggregate-reducing effect (FIG. 13).

Methods. Monoclonal antibodies recognizing different epitopes of theα-synuclein molecule and an irrelevant, isotype-matched control antibodywere dissolved in sterile phosphate-buffered-saline solution forinjection into mice (Table 6). The animals used were 4 to 8 month-oldheterozygous transgenic mice overexpressing human wildtype α-synucleinin the brain under the transcriptional control of the PDGF promoter.From 4 to 6 different transgenic mice were used for each of theantibodies.

TABLE 6 α-Synuclein Antibodies and Controls Used for 3 IntracerebralInjection in a Transgenic Model of Neuronal Synucleopathy. Monoclonalantibody Epitope Isotype 8A5 α-synuclein C-terminus IgGl 6H7 α-synucleinN-terminus IgGl 9E4 α-synuclein aa118-126 IgGl 27/1 control IgGl

For each mouse, 2 μl of a 2 mg/ml antibody solution were injected foreach injection site stereotactically under anesthesia into the deeplayers of the parietal neocortex of the right brain hemisphere(ipsilateral side). The left hemispheres (contralateral side) served asan baseline control for each mouse. The three stereotactic injectioncoordinates were: injection 1: 2.0 mm Bregma, 1.5 mm Lateral, 2.0 mmDepth; injection 2: 0.4 mm Bregma, 1.5 mm Lateral, 1.4 mm Depth;injection 3: −2.3 mm Bregma, 1.5 mm Lateral, 1.2 mm Depth). Injectionsites were sutured and mice were monitored until they recovered fromanesthesia. The investigator performing the injections was blinded as towhich antibody was injected each time. Two weeks after injection, micewere euthanized following institutional guidelines. Their brains wereremoved, fixed in 4% paraformaldehyde for 48 h, and cut coronally at 40μm thickness using a Leica vibratome. Every third section throughout thebrain for each animal was stained by immunoperoxidase staining with apolyclonal α-synuclein antibody (ELADW-47, recognizing α-synuclein aminoacids 115-122). Based on location for each injection site, two-foursections around each of the three injection site were selected for eachanimal. Intraneuronal α-synuclein aggregates were counted in 4microscopic fields (20× objective) around the injection site in theipsilateral hemisphere, and in 4 fields corresponding fields in thecontralateral control hemisphere. The α-synuclein aggregate counts forall sections counted (3-12 sections total/animal) were summed for eachhemisphere. Finally, for each animal the difference between the totalα-synuclein aggregate count between the two hemisphere was calculatedand expressed as % difference between the contralateral and theipsilateral hemisphere, thus providing a measure of the effect ofα-synuclein antibodies on aggregate clearance for each individual mouse.Sections were blind-coded and the code was broken when analysis wascomplete.

Example XI: A Monoclonal Antibody Against the c-Terminus ofAlpha-Synuclein Enters the Brain and Recognizes Alpha-Synuclein

Human α-syn tg mice were intravenously injected with 9E4-FITC at adosage of 5 mg/kg; 100 The mice were euthanized one week later todetermine the presence of the antibody in the brain and CSF. Wevisualized the fluorescent antibody directly, and found that theantibody did, in fact, traffic into the brain and specificallyrecognized asyn in the neuronal cell body and synapses. Aperipherally-delivered control IgG showed only backgroundimmunoreactivity in both tg and nontg mice. Furthermore, we found thatCSF from treated mice was immunoreactive against asyn in the brains ofuntreated tg mice, and showed a pattern of staining similar to directimmunolabeling with the 9E4-FITC antibody. (See FIG. 14.)

Example XII: Passive Immunization with an Antibody Against C-Terminalα-Syn Reduces α-Syn Deficits in Tg Mice

The objective of the present study was to show passive immunization withan antibody against α-syn C-terminus traffics into the CNS and iscapable of recognizing and clearing α-syn aggregates in the brain of tgmice. For this purpose human α-syn tg mice were injectedintraperitoneally with 9E4 at a dosage of of 1 mg/kg for six months.Animals treated with 9E4 were euthanized after 6 months of weeklyinjections. Reduced levels of α-syn oligomers and insoluble forms ofα-syn were found in the brains of injected tg mice. (See FIG. 15.)

Example XIII: Immunization with an Antibody Against the c-Terminus ofα-Synuclein Promotes Clearance Via Activation of the Autophagy Pathwayin a Transgenic of Parkinson's Disease

A monoclonal antibody (clone 9E4) tagged with FITC was injected IV innontg and alpha-syn tg mice. After 3 days post injection low levels ofthe antibody were detected in the brain, while high levels were detectedin plasma. At 14 and 30 days post injection higher levels were detectedin the brain with decreasing levels in the plasma as evidenced both byimmunocytochemistry and ELISA. In the brains of tg mice, the 9E4-FITCantibody was detected in association with granular α-syn aggregates inneurons. These aggregates were co-labeled with antibodies againstlysosomal markers (cathepsin D). Control experiments with a non-immuneIgG-FITC show only background labeling in nontg and alpha-syn tg mice.Moreover, immunolabeling of sections from alpha-syn tg mice with the CSFof mice treated with 9E4-FITC immunolabeled synapses and neurons insections from untreated alpha-syn tg mice, in contrast no labeling wasobserved with the CSF of mice treated with non-immune IgG-FITC.

Clearance of alpha-syn in the tg mice immunized with 9E4 was probablyrelated to activation of the autophagy pathway because levels ofbeclin-1, LC3 cleavage and Atg5 were increased and co-localized with theα-syn aggregates in the neurons of immunized tg mice. (See FIG. 16.)These results suggest that a monoclonal antibody against an epitope ator near the c-terminus of α-syn traffics into the CNS, recognizesaggregates in affected neurons and t triggers clearance via a lysosomalpathway, such as autophagy. FIG. 17 illustrates such pathways. In theleft of the figure, the antibody binds to membrane-bound alpha synucleinfrom outside the cells, and the resulting complex is endocytosed andcombines with a lysosome inside the cell forming an autophagosome andtriggering degradation by lysosomal enzymes. In the right of the figure,alpha-synuclein and the antibody to alpha-synuclein are separatelyendocytosed, the antibody likely gaining entry via Fc receptors, andcombined with a lysosome in the cell, followed by lysosomal degradation.

Deposit

The following monoclonal antibody-producing cell lines have beendeposited under the provisions of the Budapest Treaty with the AmericanType Culture Collection (ATCC, P.O. Box 1549, Manassas, Va. 20108) onthe dates indicated:

Monoclonal Epitope/ Date of Accession antibody Cell Line SpecificityIsotype Deposit No. 11A5 JH22.11A5.6.29.70.54.16.14 alpha-synuclein IgGlFeb. 26, 2007 PTA-8222 residues 124-134 phospoSER129 8A5 JH4.8A5.25.7.36alpha-synuclein IgGl Aug. 4, 2005 PTA-6909 C-terminus 1H7JH17.1H7.4.24.34 alpha-synuclein IgGl Feb. 26, 2007 PTA-8220 residues91-99 9E4 JH7.9E4.3.37.1.14.2 alpha-synuclein IgG1 Feb. 26, 2007PTA-8221 residues 118-126 6H7 JH17.6H7.1.54.28 alpha-synuclein IgGl Aug.4, 2005 PTA-6910 N-terminus

It will be apparent to one of ordinary skill in the art that manychanges and modifications can be made thereto without departing from thespirit or scope of the appended claims. Unless otherwise apparent fromthe context, any step, feature, embodiment, or aspect can be used incombination with any other. All publications and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

1-32. (canceled)
 33. A method of obtaining a human antibody binding toan epitope within a defined segment of alpha synuclein comprisingscreening a DNA library obtained from B cells of a human for expressionof antibodies binding to human alpha synuclein and further screening theantibodies against deletion mutants of human alpha synuclein to identifyan antibody binding within residues 1-10 or 110-130 of alpha synuclein.34. The method of claim 33, wherein the antibody binds within residues1-10 of alpha synuclein.
 35. The method of claim 33, wherein theantibody binds within residues 110-130 of alpha synuclein.
 36. Themethod of claim 33, wherein the antibody binds within residues 118-126of alpha synuclein.
 37. The method of claim 33, further comprisingtransforming nucleic acids encoding heavy and light of the antibody intoa host cell and expressing the antibody.
 38. The method of claim 37,wherein the host cell is a CHO cell.
 39. The method of claim 37, furthercomprising purifying the antibody from a culture of the host cell. 40.The method of claim 37, further comprising incorporating the antibodyinto a pharmaceutical composition.